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Instruction Manual
MultiSite Gateway(R) ThreeFragment Vector Construction Kit
Using Gateway(R) Technology to simultaneously clone multiple DNA fragments
Catalog no. 12537-023
Version C 29 November 2004 25-0541 A Limited Use Label License covers this product (see Purchaser Notification). By use of this product, you accept the terms and conditions of the Limited Use Label License.
ii
Table of Contents
Table of Contents ..................................................................................................................................................... iii MultiSite Gateway(R) BP and LR Recombination Reaction Protocols for Experienced Users ......................... v Kit Contents and Storage....................................................................................................................................... vii Accessory Products ................................................................................................................................................. ix
Introduction ......................................................................................................................1
Overview ................................................................................................................................................................... 1 The Gateway(R) Technology ...................................................................................................................................... 2 MultiSite Gateway(R) Technology............................................................................................................................. 3 MultiSite Gateway(R) BP Recombination Reactions............................................................................................... 6 Features of the MultiSite Gateway(R) Vectors ......................................................................................................... 9
Methods...........................................................................................................................10
Propagating the MultiSite Gateway(R) Vectors..................................................................................................... 10 Types of Entry Clones ............................................................................................................................................ 11 Designing attB PCR Primers ................................................................................................................................. 14 Producing attB PCR Products ............................................................................................................................... 17 Purifying attB PCR Products................................................................................................................................. 18 Creating Entry Clones Using the BP Recombination Reaction ........................................................................ 19 Performing the BP Recombination Reaction....................................................................................................... 24 Transforming One Shot(R) TOP10 Competent Cells ............................................................................................ 27 Sequencing Entry Clones....................................................................................................................................... 30 Creating Expression Clones Using the MultiSite Gateway(R) LR Recombination Reaction........................... 31 Performing the MultiSite Gateway(R) LR Recombination Reaction................................................................... 34 Troubleshooting...................................................................................................................................................... 37
Appendix .........................................................................................................................41
Map of pDONRTMP4-P1R........................................................................................................................................ 41 Map of pDONRTM221 .............................................................................................................................................. 42 Map of pDONRTMP2R-P3........................................................................................................................................ 43 Features of pDONRTM Vectors ............................................................................................................................... 44 Map and Features of pDESTTMR4-R3 .................................................................................................................... 45 Map of pMS/GW.................................................................................................................................................... 47 Technical Service .................................................................................................................................................... 48 Purchaser Notification ........................................................................................................................................... 50 Gateway(R) Clone Distribution Policy.................................................................................................................... 52 Product Qualification............................................................................................................................................. 53 Glossary of Terms................................................................................................................................................... 54 References ................................................................................................................................................................ 56
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MultiSite Gateway(R) BP and LR Recombination Reaction Protocols for Experienced Users
Introduction
This quick reference sheet is provided for experienced users of the MultiSite Gateway(R) Technology. If you are performing the BP or MultiSite Gateway(R) LR recombination reactions for the first time, we recommend following the detailed protocols provided in the manual.
BP Recombination Perform a BP recombination reaction between each attB-flanked DNA fragment and the appropriate attP-containing donor vector to generate an entry clone (see page 19 Reaction
for details). 1. Add the following components to a 1.5 ml microcentrifuge tube at room temperature and mix: attB PCR product (20-50 fmoles) pDONR vector (supercoiled, 150 ng/l) TE Buffer, pH 8.0 2. 3. 4. 5.
TM
1-7 l 1 l to 8 l
Vortex BP ClonaseTM II enzyme mix briefly. Add 2 l to the components above and mix well by vortexing briefly twice. Incubate reaction at 25C for 1 hour. Add 1 l of 2 g/l Proteinase K solution and incubate at 37C for 10 minutes. Transform 1 l of the reaction into competent E. coli and select for kanamycinresistant entry clones.
MultiSite Gateway(R) LR Recombination Reaction
Perform a MultiSite Gateway(R) LR recombination reaction between multiple entry clones (attL4-5 element-attR1 + attL1-gene of interest-attL2 + attR2-3 element-attL3) and the pDESTTMR4-R3 vector to generate an expression clone (attB4-5 elementattB1-gene of interest-attB2-3 element-attB3). 1. Add the following components to a 1.5 ml microcentrifuge tube at room temperature and mix: Entry clones (supercoiled, 20-25 fmoles each) pDESTTMR4-R3 (supercoiled, 60 ng/l) 5X LR Clonase Plus reaction buffer TE Buffer, pH 8.0 2. 3. 4. 5.
TM TM
1-11 l 1 l 4 l to 16 l
Vortex LR Clonase Plus enzyme mix briefly. Add 4 l to the components above and mix well by vortexing briefly twice. Incubate reaction at 25C for 16 hours (or overnight). Add 2 l of 2 g/l Proteinase K solution and incubate at 37C for 10 minutes. Transform 2 l of the reaction into competent E. coli and select for ampicillinresistant expression clones.
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Kit Contents and Storage
Shipping/Storage
The MultiSite Gateway(R) Three-Fragment Vector Construction Kit is shipped on dry ice in four boxes as described below. Upon receipt, store each box as detailed below. Box 1 2 3 4 Vectors BP Clonase II Enzyme Mix LR Clonase Plus Enzyme Mix One Shot TOP10 Chemically Competent E. coli
(R) TM TM
Item
Storage -20C -20C -80C -80C
Vectors
The Vectors box (Box 1) contains the following items. Store Box 1 at -20C. Item pDONRTMP4-P1R pDONR P2R-P3 pDONR 221 pDEST R4-R3 pMS/GW control plasmid
TM TM TM
Composition Lyophilized in TE Buffer, pH 8.0 Lyophilized in TE Buffer, pH 8.0 Lyophilized in TE Buffer, pH 8.0 Lyophilized in TE Buffer, pH 8.0 Lyophilized in TE Buffer, pH 8.0
Amount 6 g 6 g 6 g 6 g 10 g
BP ClonaseTM II Enzyme Mix
The following reagents are supplied with the BP ClonaseTM II enzyme mix (Box 2). Store Box 2 at -20C for up to 6 months. For long-term storage, store at -80C. Item BP ClonaseTM II Enzyme Mix Proteinase K solution 2 g/l in: 10 mM Tris-HCl, pH 7.5 20 mM CaCl2 50% glycerol 30% PEG/Mg solution pEXP7-tet positive control 30% PEG 8000/30 mM MgCl2 50 ng/l in TE Buffer, pH 8.0 1 ml 20 l Composition Proprietary Amount 40 l 40 l
continued on next page
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Kit Contents and Storage, continued
LR ClonaseTM Plus Enzyme Mix
The following reagents are supplied with the LR ClonaseTM Plus enzyme mix (Box 3). Store Box 3 at -80C. Item LR Clonase Plus Enzyme Mix 5X LR Clonase Plus Reaction Buffer Proteinase K solution
TM TM
Composition Proprietary Proprietary 2 g/l in: 10 mM Tris-HCl, pH 7.5 20 mM CaCl2 50% glycerol
Amount 80 l 100 l 40 l
One Shot(R) TOP10 Reagents
The One Shot(R) TOP10 Chemically Competent E. coli kit (Box 4) contains the following reagents. Transformation efficiency is 1 x 109 cfu/g DNA. Store Box 4 at -80C. Item S.O.C. Medium (may be stored at room temperature or +4C) Composition 2% tryptone 0.5% yeast extract 10 mM NaCl 2.5 mM KCl 10 mM MgCl2 10 mM MgSO4 20 mM glucose TOP10 chemically competent cells pUC19 Control DNA -10 pg/l in 5 mM Tris-HCl, 0.5 mM EDTA, pH 8 21 x 50 l 50 l Amount 6 ml
Genotype of TOP10
Note that this strain cannot be used for single-strand rescue of DNA. F- mcrA (mrr-hsdRMS-mcrBC) 80lacZM15 lac74 recA1 araD139 (ara-leu)7697 galU galK rpsL (StrR) endA1 nupG
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Accessory Products
Introduction
The products listed in this section may be used with the MultiSite Gateway(R) ThreeFragment Vector Construction Kit. For more information, refer to our Web site (www.invitrogen.com) or call Technical Service (see page 48).
Additional Products
Many of the reagents supplied in the MultiSite Gateway(R) Three-Fragment Vector Construction Kit as well as other products suitable for use with the kit are available separately from Invitrogen. Ordering information for these reagents is provided below. Item BP Clonase II Enzyme Mix LR Clonase Plus Enzyme Mix Library Efficiency DH5 Chemically Competent Cells One Shot(R) TOP10 Chemically Competent E. coli One Shot(R) ccdB Survival T1R Chemically Competent E. coli pDONRTM221 M13 Forward (-20) Sequencing Primer M13 Reverse Sequencing Primer S.N.A.P. MiniPrep Kit S.N.A.P. MidiPrep Kit S.N.A.P. Gel Purification Kit Ampicillin Kanamycin Sulfate Platinum Pfx DNA Polymerase Platinum Taq DNA Polymerase High Fidelity Dpn I REact 4 Buffer
(R) (R) (R) TM TM TM TM TM TM
Quantity 20 reactions 100 reactions 20 reactions 5 x 0.2 ml 20 x 50 l 20 x 50 l 6 g 2 g 2 g 100 reactions 20 reactions 25 reactions 20 ml (10 mg/ml) 100 ml (10 mg/ml) 100 reactions 250 reactions 100 reactions 500 reactions 100 units 2 x 1 ml
Catalog no. 11789-020 11789-100 12538-013 18263-012 C4040-03 C7510-03 12536-017 N520-02 N530-02 K1900-01 K1910-01 K1999-25 11593-019 15160-054 11708-013 11708-021 11304-011 11304-029 15242-019 16304-016 continued on next page
ix
Accessory Products, continued
Gateway(R) Entry Vectors
The MultiSite Gateway(R) Three-Fragment kit provides the pDONRTM221 vector to facilitate creation of attL1 and attL2-flanked entry clones. Alternatively, a variety of Gateway(R) entry vectors are available from Invitrogen to allow creation of entry clones using TOPO(R) Cloning or restriction digestion and ligation. For more information about the various entry vectors and their features, see our Web site (www.invitrogen.com) or contact Technical Service (see page 48). Item pENTR /D-TOPO Cloning Kit
TM (R)
Quantity 20 reactions 480 reactions 500 reactions
Catalog no. K2400-20 K2400-480 K2400-500 K2420-20 K2420-480 K2420-500 11813-011 11816-014 11817-012 11818-010 11819-018
pENTR /SD/D-TOPO Cloning Kit
TM
(R)
20 reactions 480 reactions 500 reactions
pENTR 1A pENTRTM2B pENTRTM3C pENTR 4 pENTR 11
TM TM
TM
10 g 10 g 10 g 10 g 10 g
x
Introduction Overview
Introduction
The MultiSite Gateway(R) Three-Fragment Vector Construction Kit facilitates rapid and highly efficient construction of an expression clone containing your choice of promoter, gene of interest, and termination or polyadenylation sequence. Other sequences of interest may be easily substituted or incorporated, providing added flexibility for your vector construction needs. Based on the Gateway(R) Technology (Hartley et al., 2000), the MultiSite Gateway(R) Technology uses site-specific recombinational cloning to allow simultaneous cloning of multiple DNA fragments in a defined order and orientation. For more information about the Gateway(R) Technology, see the next page.
Important
The MultiSite Gateway(R) Three-Fragment Vector Construction Kit is designed to help you create a multiple-fragment clone or an expression clone using the MultiSite Gateway(R) Technology. Although the kit has been designed to help you produce your expression clone in the simplest, most direct fashion, use of the kit is geared towards those users who are familiar with the concepts of the Gateway(R) Technology and site-specific recombination. A working knowledge of the Gateway(R) Technology is recommended. A brief overview about the Gateway(R) Technology is provided in this manual. For more details about the Gateway(R) Technology and the recombination reactions, refer to the Gateway(R) Technology with ClonaseTM II manual. The manual is available for downloading from our Web site (www.invitrogen.com) or by calling Technical Service (see page 48).
Purpose of This Manual
This manual provides an overview of the MultiSite Gateway(R) Technology, and provides instructions and guidelines to: 1. 2. 3. Design three sets of forward and reverse attB PCR primers, and amplify your three DNA sequences of interest. Perform a BP recombination reaction with each attB PCR product and a specific donor vector to generate three types of entry clones. Perform a MultiSite Gateway(R) LR recombination reaction with your three entry clones and the pDESTTMR4-R3 destination vector to generate an expression clone which may then be used in the appropriate application or expression system.
Glossary of Terms
To help you understand the terminology used in the MultiSite Gateway(R) Technology, a glossary of terms is provided in the Appendix, page 54 for your convenience.
1
The Gateway(R) Technology
Gateway(R) Technology
The Gateway(R) Technology is a universal cloning method based on the bacteriophage lambda site-specific recombination system (Landy, 1989; Ptashne, 1992) that provides a rapid and highly efficient way to transfer heterologous DNA sequences into multiple vector systems for functional analysis and protein expression (Hartley et al., 2000).
Lambda Recombination Reactions
In lambda, recombination occurs between lambda and the E. coli chromosome via specific recombination sequences (att sites), and is catalyzed by a mixture of recombination proteins (ClonaseTM II enzyme mix). The reactions are described in the table below. Pathway Lysogenic Lytic Reaction attB x attP attL x attR attL x attR attB x attP Catalyzed by... BP ClonaseTM II (Int, IHF) LR ClonaseTM II (Int, Xis, IHF)
Gateway(R) Recombination Reactions
The Gateway(R) Technology uses modified and optimized att sites to permit transfer of heterologous DNA sequences between vectors. Two recombination reactions constitute the basis of the Gateway(R) Technology: * BP Reaction: Recombination of an attB substrate (e.g. attB PCR product or expression clone) with an attP substrate (donor vector) to create an attLcontaining entry clone (see diagram below). The reaction is catalyzed by BP ClonaseTM II enzyme mix, a mixture of the Integrase (Int) and E. coli Integration Host Factor (IHF) proteins.
att P
ccdB
att B
gene
att B
att P BP Clonase II
att L
gene
att L
att R
ccdB
att R
attB-flanked PCR product or attB expression clone
donor vector
entry clone
by-product
*
LR Reaction: Recombination of an attL-containing entry clone with an attRcontaining destination vector to create an attB-containing expression clone (see diagram below). The reaction is catalyzed by LR ClonaseTM II enzyme mix, a mixture of the Int and Excisionase (Xis) proteins, and the E. coli IHF protein.
att R
ccdB
att L
gene
att L
att R
LR Clonase II
att B
gene
att B
att P
ccdB
att P
entry clone
destination vector
expression clone
by-product
For More Information
For details about the Gateway(R) Technology, lambda DNA recombination, att sites, and the BP and LR recombination reactions, refer to the Gateway(R) Technology with ClonaseTM II manual. This manual is available for downloading from our Web site (www.invitrogen.com) or by calling Technical Service (see page 48).
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MultiSite Gateway(R) Technology
Introduction
The MultiSite Gateway(R) Three-Fragment Vector Construction Kit uses modifications of the site-specific recombination reactions of the Gateway(R) Technology (see the next page for more information) to allow simultaneous cloning of three DNA fragments in a defined order and orientation to create your own expression clone. To generate your own expression clone, you will: 1. Amplify your three DNA sequences of interest (i.e. 5 element, gene of interest, and 3 element) using the recommended attB primers to generate PCR products that are flanked by attB sites. To ensure that your fragments are joined in a specific order, specific attB sites must flank each PCR product. Use the PCR products in separate BP recombination reactions with three donor vectors (pDONRTMP4-P1R, pDONRTM221, pDONRTMP2R-P3) to generate three entry clones containing your DNA sequences of interest. For more information about the donor vectors, see page 20. Use the three entry clones in a single MultiSite Gateway(R) LR recombination reaction with a specially designed destination vector, pDESTTMR4-R3, to create your expression clone of interest (see the diagram below). For more information about pDESTTMR4-R3, see page 32.
ori kan
ori
2.
3.
kan
pENTR-5 element
attL4
pENTR-3 element
5 element
attR1
attR2
3 element
attL3
Entry Clones
attL1
gene
attL2
pENTR-gene
ori kan
Destination Vector
attR4
ccdB
TM
CmR
attR3
pDEST R4-R3 ori
amp
LR ClonaseTM Plus
attB4 5 element attB1 gene attB2 3 element attB3
Expression Clone
Your expression clone
ori
amp
continued on next page
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MultiSite Gateway(R) Technology, continued
Modifications to the att Sites
To permit recombinational cloning using the Gateway(R) Technology, the wild-type att sites have been modified to improve the efficiency and specificity of the Gateway(R) BP and LR recombination reactions (see the Gateway(R) Technology manual for details). In the MultiSite Gateway(R) System, the att sites have been optimized further to accommodate simultaneous, recombinational cloning of multiple DNA fragments. These modifications include alterations to both the sequence and length of the att sites, resulting in the creation of "new" att sites exhibiting enhanced specificities and the improved efficiency required to clone multiple DNA fragments in a single reaction. In the MultiSite Gateway(R) Three-Fragment kit, four att sites are used versus two att sites in the standard Gateway(R) Technology. For example, four attB sites are used in the MultiSite Gateway(R) Three-Fragment kit (see table below). Various combinations of these attB sites will flank each PCR product containing your DNA fragment of interest. MultiSite Gateway(R) attB1 attB2 attB3 attB4 Gateway(R) attB1 attB2
Specificity of the Modified att Sites
In general, the modified att sites in the MultiSite Gateway(R) Technology demonstrate the same specificity as in the Gateway(R) Technology. That is: * * attB sites react only with attP sites; similarly attB1 sites react only with attP1 sites to generate attL1 sites attL sites react only with attR sites; similarly attL1 sites react only with attR1 sites to generate attB1 sites
However, depending on the orientation and position of the attB site and attP site in relation to the DNA fragment of interest or the donor vector, respectively, performing the BP recombination reaction can result in creation of an attR site instead of an attL site. Specifically: * * attB1 sites react with attP1R sites to generate attR1 sites attB2 sites react with attP2R sites to generate attR2 sites
See the next page for an example. See the next section, pages 6-8 for diagrams of these BP recombination reactions. continued on next page
4
MultiSite Gateway(R) Technology, continued
Example
In this example, an attB4 and attB1-flanked PCR product is used in a BP recombination reaction with pDONRTMP4-P1R. attB4-PCR product-attB1 x pDONRTMP4-P1R attL4-PCR product-attR1 Because of the orientation and position of the attB1 and attP1R site in the PCR product and donor vector, respectively, the resulting entry clone contains the PCR product flanked by an attL4 site and an attR1 site rather than two attL sites. See page 6 for a diagram of this BP recombination reaction.
MultiSite Gateway(R) Donor Vectors
The MultiSite Gateway(R) donor vectors are used to clone attB-flanked PCR products to generate entry clones, and contain similar elements as other Gateway(R) donor vectors. However, because your PCR products will be flanked by different attB sites, three different donor vectors are required to facilitate generation of the three types of entry clones required for MultiSite Gateway(R): * * * pDONRTMP4-P1R: Use to clone attB4 and attB1-flanked PCR products. pDONRTM221: Use to clone attB1 and attB2-flanked PCR products. pDONRTMP2R-P3: Use to clone attB2 and attB3-flanked PCR products.
For more information about the general features of the donor vectors, see page 9. For a map and a description of the features of each pDONRTM vector, see the Appendix, pages 41-44.
Note: While pDONRTM221 may be used in standard Gateway(R) reactions, the pDONRTMP4P1R and pDONRTMP2R-P3 vectors may only be used for MultiSite Gateway(R) applications.
The MultiSite Gateway(R) destination vector, pDESTTMR4-R3, is designed for use in MultiSite (R) the MultiSite Gateway(R) three-fragment LR recombination reaction with the three Gateway Destination Vector entry clones described above. The pDESTTMR4-R3 vector contains attR4 and attR3 sites flanking a selection cassette and allows generation of the expression clone of interest. Note that other Gateway(R) destination vectors are not suitable for use in the MultiSite Gateway(R) LR reaction. For more information about the general features of the destination vector, see page 9. For a map and a description of the features of the pDESTTMR4-R3 vector, see the Appendix, pages 45-46.
LR ClonaseTM Plus Enzyme Mix
The MultiSite Gateway(R) LR recombination reaction is catalyzed by an optimized LR ClonaseTM, LR ClonaseTM Plus enzyme mix. LR ClonaseTM Plus enzyme mix facilitates efficient recombinational cloning of multiple DNA fragments, but may also be used in the standard Gateway(R) LR recombination reaction. Note that LR ClonaseTM enzyme mix is not suitable for use in the MultiSite Gateway(R) LR recombination reaction.
5
MultiSite Gateway(R) BP Recombination Reactions
Introduction
The MultiSite Gateway(R) BP recombination reaction facilitates production of entry clones from your three attB-flanked PCR products. Since each PCR product is flanked by a specific combination of attB sites, specific donor vectors must also be used. An illustration of each BP recombination reaction is provided in this section.
Important
Note that the att sites used in MultiSite Gateway(R) have been optimized to improve specificity and efficiency of the MultiSite Gateway(R) LR recombination reaction, and may vary in size and sequence from those used in the Gateway(R) Technology.
attB 5 Element x pDONRTMP4-P1R Recombination Region
The diagram below depicts the recombination reaction between the attB4 and attB1-flanked PCR product (i.e. attB 5 element) and pDONRTMP4-P1R to create an entry clone and a by-product. Features of the Recombination Region: * Shaded regions correspond to those sequences transferred from the attB 5 element into the entry clone following recombination. Note that attL4 and attR1 sites flank the 5 element in the entry clone. Boxed regions correspond to those sequences transferred from the donor vector into the by-product following recombination.
5 Element
*
attB5 Element
GGGGACAACTTTGTATAGAAAAGTTG--------------CAAGTTTGTACAAAAAAGCAGTCCCC CCCCTGTTGAAACATATCTTTTCAAC--------------GTTCAAACATGTTTTTTCGTCAGGGG attB4
X
attB1
pDONR P4-P1R
vector---N75-CAACTTTGTATAGAAAAGTTG-N136--------------N75-CAACTTTGTACAAAAAAGTTG-N136---vector ccdB-CmR vector---N75-GTTGAAACATATCTTTTCAAC-N136--------------N75-GTTGAAACATGTTTTTTCAAC-N136---vector attP4 attP1R
BP ClonaseTM
Entry clone
vector---N75-CAACTTTGTATAGAAAAGTTG--------------CAAGTTTGTACAAAAAAGTTG-N136---vector 5 Element vector---N75-GTTGAAACATATCTTTTCAAC--------------GTTCAAACATGTTTTTTCAAC-N136---vector attL4
+
attR4
attR1
By-product
GGGGACAACTTTGTATAGAAAAGTTG-N136--------------N75-CAACTTTGTACAAAAAAGCAGTCCCC ccdB-CmR CCCCTGTTGAAACATATCTTTTCAAC-N136--------------N75-GTTGAAACATGTTTTTTCACCAGGGG attL1
continued on next page
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MultiSite Gateway(R) BP Recombination Reactions, continued
attB Gene x pDONRTM221 Recombination Region
The diagram below depicts the recombination reaction between the attB1 and attB2-flanked PCR product (i.e. attB gene) and pDONRTM221 to create an entry clone and a by-product. Features of the Recombination Region: * Shaded regions correspond to those sequences transferred from the attB PCR product into the entry clone following recombination. Note that the PCR product in the entry clone is flanked by attL1 and attL2 sites, and is suitable for use in all standard Gateway(R) applications. Boxed regions correspond to those sequences transferred from the donor vector into the by-product following recombination.
GENE
*
attB-gene
GGGGACAAGTTTGTACAAAAAAGCAGGCT--------------ACCCAGCTTTCTTGTACAAAGTGGTCCCC CCCCTGTTCAAACATGTTTTTTCGTCCGA--------------TGGGTCGAAAGAACATGTTTCACCAGGGG attB1
X
attB2
pDONRTM221
vector---N75-CCAACTTTGTACAAAAAAGCTGAAC-N100--------------N100-GTTCAGCTTTCTTGTACAAAGTTGG-N75---vector ccdB-CmR vector---N75-GGTTGAAACATGTTTTTTCGACTTG-N100--------------N100-CAAGTCGAAAGAACATGTTTCAACC-N75---vector attP1 attP2
BP ClonaseTM
Entry clone
vector---N75-CCAACTTTGTACAAAAAAGCAGGCT------------------ACCCAGCTTTCTTGTACAAAGTTGG-N75---vector GENE vector---N75-GGTTGAAACATGTTTTTTCGTCCGA------------------TGGGTCGAAAGAACATGTTTCAACC-N75---vector attL1
+
attL2
By-product
GGGGACAAGTTTGTACAAAAAAGCTGAAC-N100--------------N100-GTTCAGCTTTCTTGTACAAAGTGGTCCCC ccdB-CmR CCCCTGTTCAAACATGTTTTTTCGACTTG-N100--------------N100-CAAGTCGAAAGAACATGTTTCACCAGGGG attR1 attR2
continued on next page
7
MultiSite Gateway(R) BP Recombination Reactions, continued
attB 3 Element x pDONRTMP2R-P3 Recombination Region
The diagram below depicts the recombination reaction between the attB2 and attB3-flanked PCR product (i.e. attB 3 element) and pDONRTMP2R-P3 to create an entry clone and a by-product. Features of the Recombination Region: * Shaded regions correspond to those sequences transferred from the attB 3 element into the entry clone following recombination. Note that attR2 and attL3 sites flank the 3 element in the entry clone. Boxed regions correspond to those sequences transferred from the donor vector into the by-product following recombination.
3 Element
*
attB3 Element
GGGGACAGCTTTCTTGTACAAAGTGG--------------CAACTTTATTATACAAAGTTGTCCCC CCCCTGTCGAAAGAACATGTTTCACC--------------GTTGAAATAATATGTTTCAACAGGGG attB2
X
ccdB-CmR
attB3
pDONR P2R-P3
vector---N136-CAACTTTCTTGTACAAAGTTG-N75--------------N136-CAACTTTATTATACAAAGTTG-N75---vector
vector---N136-GTTGAAAGAACATGTTTCAAC-N75--------------N136-GTTGAAATAATATGTTTCAAC-N75---vector attP2R ATTP3
BP ClonaseTM
Entry clone
vector---N136-CAACTTTCTTGTACAAAGTGG--------------CAACTTTATTATACAAAGTTG-N75---vector 3 Element vector---N136-GTTGAAAGAACATGTTTCACC--------------GTTCAAATAATATGTTTCAAC-N75---vector attR2
+
attL2
attL3
By-product
GGGGACAGCTTTCTTGTACAAAGTTG-N75--------------N136-CAACTTTATTATACAAAGTTGTCCCC ccdB-CmR CCCCTGTCGAAAGAACATGTTTCAAC-N75--------------N136-GTTGAAACATGTTTTTTCAACAGGGG attR3
8
Features of the MultiSite Gateway(R) Vectors
MultiSite Gateway(R) Vectors
Two types of MultiSite Gateway(R)-adapted vectors are available from Invitrogen: Gateway(R) Vector Donor vector (pDONR )
TM
Characteristics Contains attP sites Used to clone attB-flanked PCR products to generate entry clones
Destination vector
Contains attR sites Recombines with multiple entry clones in a MultiSite Gateway(R) LR reaction to generate an expression clone
Common Features of the MultiSite Gateway(R) Vectors
To enable recombinational cloning and efficient selection of entry or expression clones, the MultiSite Gateway(R) donor and destination vectors contain two att sites flanking a cassette containing: * * The ccdB gene (see below) for negative selection Chloramphenicol resistance gene (CmR) for counterselection
After a BP or MultiSite Gateway(R) LR recombination reaction, this cassette is replaced by the gene of interest to generate the entry clone and expression clone, respectively.
ccdB Gene
The presence of the ccdB gene allows negative selection of the donor and destination vectors in E. coli following recombination and transformation. The ccdB protein interferes with E. coli DNA gyrase (Bernard and Couturier, 1992), thereby inhibiting growth of most E. coli strains (e.g. TOP10, DH5TM). When recombination occurs (i.e. between a destination vector and an entry clone or between a donor vector and an attB PCR product), the gene of interest replaces the ccdB gene. Cells that take up unreacted vectors carrying the ccdB gene or by-product molecules retaining the ccdB gene will fail to grow. This allows high-efficiency recovery of the desired clones.
9
Methods Propagating the MultiSite Gateway(R) Vectors
Introduction
The MultiSite Gateway(R) Three-Fragment Vector Construction Kit includes the following vectors. See the guidelines below to propagate and maintain these vectors. Donor Vectors: * * * * * pDONRTMP4-P1R pDONRTM221 pDONRTMP2R-P3 pDESTTMR4-R3 pMS/GW
Destination Vector: Control Vector:
Propagating Donor and Destination Vectors
If you wish to propagate and maintain the pDONRTMP4-P1R, pDONRTM221, pDONRTMP2R-P3, and pDESTTMR4-R3 vectors prior to recombination, we recommend using One Shot(R) ccdB Survival T1R Chemically Competent E. coli (Catalog no. C7510-03) from Invitrogen for transformation. The ccdB Survival T1R E. coli strain is resistant to CcdB effects and can support the propagation of plasmids containing the ccdB gene. To maintain the integrity of the vector, select for transformants as follows: * * For pDONRTM vectors, use LB plates containing 50 g/ml kanamycin and 1530 g/ml chloramphenicol. For the pDESTTMR4-R3 vector, use LB plates containing 100 g/ml ampicillin and 15-30 g/ml chloramphenicol.
Note: Do not use general E. coli cloning strains including TOP10 or DH5TM for propagation and maintenance as these strains are sensitive to ccdB effects.
pMS/GW Vector
To propagate and maintain the pMS/GW plasmid, you may use any recA, endA E. coli strain including TOP10, DH5, or DH10B for transformation. We recommend using the One Shot(R) TOP10 Chemically Competent E. coli included with the kit for transformation. Select for transformants in media containing 50100 g/ml ampicillin.
10
Types of Entry Clones
Introduction
To use the MultiSite Gateway(R) Three-Fragment kit to construct your own expression clone, you will create 3 types of entry clones, then use these entry clones in a MultiSite Gateway(R) LR recombination reaction with a MultiSite Gateway(R) destination vector to generate your expression clone. For proper expression of the gene of interest, these entry clones should, at a minimum, contain the sequences described below. Note: Depending on your needs or application of interest, other sequences are possible. * An attL4 and attR1-flanked entry clone containing your 5 element of interest. The 5 element typically contains promoter sequences required to control expression of your gene of interest. Other additional sequences including an N-terminal fusion tag may be added. An attL1 and attL2-flanked entry clone containing your DNA fragment of interest. This DNA fragment generally encodes the gene of interest. To obtain proper expression in the system of choice, remember to include sequences necessary for efficient translation initiation (i.e. Shine-Dalgarno, Kozak consensus sequence, yeast consensus sequence). An attR2 and attL3-flanked entry clone containing your 3 element of interest. The 3 element typically contains transcription termination sequences or polyadenylation sequences required for efficient transcription termination and polyadenylation of mRNA. Other additional sequences including a C-terminal fusion tag may be added.
*
*
For more information about how to generate each type of entry clone, see below.
Important
If you construct an expression clone containing the elements described above (i.e. promoter of choice + gene of interest + termination or polyadenylation sequence of choice), remember that this expression clone will be expressed transiently in mammalian, yeast, and insect systems, but may be expressed stably in prokaryotic systems. To perform stable expression studies in mammalian, yeast, or insect systems, include a resistance marker in one of the entry clones (generally the attR2 and attL3-flanked entry clone).
Generating attL4 and attR1-Flanked Entry Clones
To generate an attL4 and attR1-flanked entry clone containing your 5 element of interest: 1. 2. Design appropriate PCR primers and produce your attB4 and attB1-flanked PCR product. Perform a BP recombination reaction between the attB4 and attB1-flanked PCR product and pDONRTMP4-P1R to generate the entry clone (see figure below).
att B1
5 element
att B4
att P4
ccdB
att P1R
att L4 BP Clonase II
gene
att R1
attB4 and attB1flanked PCR product
pDONR P4-P1R vector
TM
5 entry clone
continued on next page 11
Types of Entry Clones, continued
Generating attR2 and attL3-Flanked Entry Clones
To generate an attR2 and attL3-flanked entry clone containing your 3 element of interest: 1. 2. Design appropriate PCR primers and produce your attB2 and attB3-flanked PCR product. Perform a BP recombination reaction between the attB2 and attB3-flanked PCR product and pDONRTMP2R-P3 to generate the entry clone (see figure below).
att B3
3 element
att B2
att P2R
ccdB
att P3
att R2 BP Clonase II
gene
att L3
attB2 and attB3flanked PCR product
pDONR P2R-P3 vector
TM
3 entry clone
Generating attL1 and attL2-Flanked Entry Clones
The attL1 and attL2-flanked entry clone contains your gene of interest and can be used with both MultiSite Gateway(R) and traditional Gateway(R) applications. This entry clone may be generated using a variety of methods (see figure below). 1. Generate a PCR product containing attB1 and attB2 sites and use this attB PCR product in a BP recombination reaction with the pDONRTM221 vector. To use this method, refer to the guidelines and instructions provided in this manual. Clone a PCR product or a restriction enzyme fragment into an entry (pENTRTM) vector (see the next page for more information). Generate or obtain a cDNA library cloned into a Gateway(R)-compatible vector (i.e. attB-containing pCMV SPORT6 or pEXP-AD502 vectors), and use the cDNA clones in a BP recombination reaction with the pDONRTM221 vector (see the Gateway(R) Technology with ClonaseTM II manual for more information).
Entry point (cDNA, genomic DNA, cDNA library, or other DNA fragment)
2. 3.
attB1 and attB1 and attB2-flanked attB2-flanked PCR product or cDNA clone attB-expression clone
restriction enzyme fragment
PCR product
Recombine with pDONRTM221 vector
Clone into pENTRTM vector
Clone into pENTR-TOPO(R) vector
Entry clone
continued on next page
12
Types of Entry Clones, continued
Entry Vectors
Many entry vectors are available from Invitrogen to facilitate generation of entry clones. The pENTR/D-TOPO(R) and pENTR/SD/D-TOPO(R) vectors allow rapid TOPO(R) Cloning of PCR products while the pENTRTM vectors allow ligasemediated cloning of restriction enzyme fragments. All entry vectors include: * * attL1 and attL2 sites to allow recombinational cloning of the gene of interest with a destination vector to produce an expression clone. A Kozak consensus sequence for efficient translation initiation in eukaryotic cells. Some entry vectors include a Shine-Dalgarno sequence (Shine and Dalgarno, 1975) for initiation in E. coli (see table below). Kanamycin resistance gene for selection of plasmid in E. coli. pUC origin for high-copy replication and maintenance of the plasmid in E coli.
* *
For more information about the features of each pENTRTM vector, see our Web site (www.invitrogen.com) or call Technical Service (see page 48). Entry Vector pENTR/D-TOPO pENTR 1A pENTR 2B pENTRTM3C pENTRTM4 pENTR 11
TM TM TM (R) (R)
Kozak * * * * * * *
Shine-Dalgarno * * * *
Catalog no. K2400-20 K2420-20 11813-011 11816-014 11817-012 11818-010 11819-018
pENTR/SD/D-TOPO
Constructing Entry Clones
To construct an entry clone using one of the pENTRTM vectors, refer to the manual for the specific entry vector you are using. All entry vector manuals are available for downloading from our Web site (www.invitrogen.com) or by calling Technical Service (see page 48).
13
Designing attB PCR Primers
Introduction
To generate PCR products suitable for use as substrates in a Gateway(R) BP recombination reaction with a donor vector, you will need to incorporate attB sites into your PCR products. To facilitate use in MultiSite Gateway(R), each PCR product must be flanked by a different combination of attB sites (see table below). Guidelines are provided below to help you design appropriate PCR primers. DNA Sequence of Interest 5 element Gene of interest 3 element Forward PCR Primer attB4 attB1 attB2 Reverse PCR Primer attB1 attB2 attB3
Designing Your PCR Primers
The design of the PCR primers to amplify your DNA sequences of interest is critical for recombinational cloning using MultiSite Gateway(R) Technology. Consider the following when designing your PCR primers: * * Sequences required to facilitate MultiSite Gateway(R) cloning. Sequences required for efficient expression of the protein of interest (i.e. promoter sequences, termination or polyadenylation sequences, ShineDalgarno or Kozak consensus sequences). Whether or not you wish your PCR product(s) to be fused in frame with any N- or C-terminal fusion tags. Note that sequences encoding the tag are generally incorporated into your PCR product as part of the 5 or 3 element.
*
Guidelines to Design the Forward PCR Primer
When designing the appropriate forward PCR primer, consider the points below. Refer to the diagram on the next page for more help. * To enable efficient MultiSite Gateway(R) cloning, the forward primer MUST contain the following structure: 1. 2. 3. Four guanine (G) residues at the 5 end followed by The 22 or 25 bp attB site followed by At least 18-25 bp of template- or gene-specific sequences
Note: If you plan to express native protein in E. coli or mammalian cells, you may want to include a Shine-Dalgarno (Shine and Dalgarno, 1975) or Kozak consensus sequence (Kozak, 1987; Kozak, 1991; Kozak, 1990), respectively, in the attB1 forward PCR primer.
*
The attB4 and attB2 sites end with a guanine (G), and the attB1 site with a thymine (T). If you wish to fuse your PCR product in frame with an N- or Cterminal tag (as appropriate), the primer must include two additional nucleotides to maintain the proper reading frame (see diagram on the next page). Note that the two additional nucleotides in the attB1 primer cannot be AA, AG, or GA because these additions will create a translation termination codon. continued on next page
14
Designing attB PCR Primers, continued
attB Forward Primers
Design each attB forward primer to contain the following recommended sequence as listed below:
5-GGGG-ACA-AGT-TTG-TAC-AAA-AAA-GCA-GGC-TNN--(template-specific sequence)-3
attB1 5-GGGG-ACA-GCT-TTC-TTG-TAC-AAA-GTG-GNN--(template-specific sequence)-3 attB2
5-GGGG-ACA-ACT-TTG-TAT-AGA-AAA-GTT-GNN--(template-specific sequence)-3 attB4
attB1 attB2 attB4
Guidelines to Design the Reverse PCR Primer
When designing your reverse PCR primer, consider the points below. Refer to the diagram below for more help. * To enable efficient MultiSite Gateway(R) cloning, the reverse primer MUST contain the following structure: 1. 2. 3. * 1. 2. * Four guanine (G) residues at the 5 end followed by The 22 or 25 bp attB site followed by 18-25 bp of template- or gene-specific sequences The attB1 and attB2 reverse primers must include one additional nucleotide to maintain the proper reading frame (see diagram below). Any in-frame stop codons between the attB sites and your gene of interest must be removed.
If you wish to fuse your PCR product in frame with an N- or C-terminal tag:
If you do not wish to fuse your PCR product in frame with a C-terminal tag, your gene of interest or the attB2 primer must include a stop codon.
attB Reverse Primers
Design each attB reverse primer to contain the following recommended sequence as listed below:
5-GGGG-AC-TGC-TTT-TTT-GTA-CAA-ACT-TGN--(template-specific sequence)-3
attB1 5-GGGG-AC-CAC-TTT-GTA-CAA-GAA-AGC-TGG-GTN--(template-specific sequence)-3 attB2
5-GGGG-AC-AAC-TTT-GTA-TAA-TAA-AGT-TGN--(template-specific sequence)-3 attB3
attB1 attB2 attB3
continued on next page
15
Designing attB PCR Primers, continued
* 50 nmoles of standard purity, desalted oligonucleotides is sufficient for most applications. Dissolve oligonucleotides to 20-50 mM in water or TE Buffer and verify the concentration before use. For more efficient cloning of large PCR products (greater than 5 kb), we recommend using HPLC or PAGE-purified oligonucleotides.
Important
* *
16
Producing attB PCR Products
DNA Templates
The following DNA templates can be used for amplification with attB-containing PCR primers: * * * * Genomic DNA mRNA cDNA libraries Plasmids containing cloned DNA sequences
Recommended Polymerases
We recommend using the following DNA polymerases available from Invitrogen to produce your attB PCR products. Other DNA polymerases are suitable. * * To generate PCR products less than 5-6 kb for use in protein expression, use Platinum(R) Pfx DNA Polymerase (Catalog no. 11708-013). To generate PCR products for use in other applications (e.g. functional analysis), use Platinum(R) Taq DNA Polymerase High Fidelity (Catalog no. 11304-011).
Producing PCR Products
Standard PCR conditions can be used to prepare attB PCR products. Follow the manufacturer's instructions for the DNA polymerase you are using, and use the cycling parameters suitable for your primers and template. Note: In general, attB sequences do not affect PCR product yield or specificity. Remove 1-2 l from each PCR reaction and use agarose gel electrophoresis to verify the quality and yield of your PCR product. If the PCR product is of the appropriate quality and quantity, proceed to Purifying attB PCR Products, next section.
Checking the PCR Product
If your PCR template is a plasmid that contains the kanamycin resistance gene, we suggest treating your PCR reaction mixture with Dpn I before purifying the attB PCR product. This treatment degrades the plasmid (i.e. Dpn I recognizes methylated GATC sites) and helps to reduce background in the BP recombination reaction associated with template contamination. Materials Needed: * * 1. 2. 3. 4. 10X REact(R) 4 Buffer (Invitrogen, Catalog no. 16304-016) Dpn I (Invitrogen, Catalog no. 15242-019) To your 50 l PCR reaction mixture, add 5 l of 10X REact(R) 4 Buffer and 5 units of Dpn I. Incubate at 37C for 15 minutes. Heat-inactivate the Dpn I at 65C for 15 minutes. Proceed to Purifying attB PCR Products, next page.
Protocol:
17
Purifying attB PCR Products
Introduction
After you have generated your attB PCR products, we recommend purifying each PCR product to remove attB primers and any attB primer-dimers. Primers and primer-dimers can recombine efficiently with the donor vector in the BP reaction and may increase background after transformation into E. coli. A protocol is provided below to purify your PCR products.
Important
Standard PCR product purification protocols using phenol/chloroform extraction followed by sodium acetate and ethanol or isopropanol precipitation are not recommended for use in purifying attB PCR products. These protocols generally have exclusion limits of less than 100 bp and do not efficiently remove large primer-dimer products.
Materials Needed
You should have the following materials on hand before beginning: * * * * Each attB PCR product (in a 50 l volume) TE Buffer, pH 8.0 (10 mM Tris-HCl, pH 8.0, 1 mM EDTA) 30% PEG 8000/30 mM MgCl2 (supplied with the kit, Box 2) Agarose gel of the appropriate percentage to resolve your attB PCR products
PEG Purification Protocol
Use the protocol below to purify attB PCR products. Note that this procedure removes DNA less than 300 bp in size. 1. 2. Add 150 l of TE, pH 8.0 to a 50 l amplification reaction containing your attB PCR product. Add 100 l of 30% PEG 8000/30 mM MgCl2. Vortex to mix thoroughly and centrifuge immediately at 10,000 x g for 15 minutes at room temperature.
Note: In most cases, centrifugation at 10,000 x g for 15 minutes results in efficient recovery of PCR products. To increase the amount of PCR product recovered, the centrifugation time may be extended or the speed of centrifugation increased.
3. 4. 5. 6.
Carefully remove the supernatant. The pellet will be clear and nearly invisible. Dissolve the pellet in 50 l of TE, pH 8.0 (to concentration > 10 ng/l). Check the quality and quantity of the recovered attB PCR product on an agarose gel. If the PCR product is suitably purified, proceed to Creating Entry Clones Using the BP Recombination Reaction, page 19. If the PCR product is not suitably purified (e.g. attB primer-dimers are still detectable), see below.
Additional Purification
If you use the procedure above and your attB PCR product is not suitably purified, you may gel purify your attB PCR product. We recommend using the S.N.A.P.TM Gel Purification Kit available from Invitrogen (Catalog no. K1999-25).
18
Creating Entry Clones Using the BP Recombination Reaction
Introduction
Once you have generated your attB PCR products, you will perform a BP reaction to transfer the DNA sequence of interest into an attP-containing donor vector to create an entry clone. To ensure that you obtain the best possible results, we suggest that you read this section and the ones entitled Performing the BP Recombination Reaction (pages 24-26) and Transforming One Shot(R) TOP10 Competent Cells (pages 27-29) before beginning.
Choosing a Donor Vector
Since different attB sites flank each attB PCR product, a specific donor vector is required for each BP recombination reaction. Refer to the table below to determine which donor vector to use in the BP recombination reaction. See the diagrams on pages 21-23 for an illustration of the recombination region of each entry clone after the BP reaction. If your PCR product contains... attB4-PCR product-attB1 attB1-PCR product-attB2 attB2-PCR product-attB3 Then use... pDONRTMP4-P1R pDONRTM221 pDONRTMP2R-P3
Experimental Outline
To generate an entry clone, you will: 1. 2. 3. Perform a BP recombination reaction using the appropriate linear attB PCR product and a supercoiled, attP-containing donor vector (see above). Transform the reaction mixture into a suitable E. coli host (see page 27). Select for entry clones.
For optimal results, perform the BP recombination reaction using:
Important
* *
Linear attB PCR products Supercoiled donor vector continued on next page
19
Creating Entry Clones Using the BP Recombination Reaction, continued
Donor Vectors
The pDONRTMP4-P1R, pDONRTM221, and pDONRTMP2R-P3 vectors are supplied with the kit to facilitate generation of entry clones using the BP recombination reaction. The donor vectors contain the following elements: * * * * * * Two attP sites for recombinational cloning of attB-containing PCR products The ccdB gene located between the attP sites for negative selection The chloramphenicol resistance gene (CmR) located between the two attP sites for counterselection M13 forward (-20) and M13 reverse primer binding sites to facilitate sequencing of the entry clone, if desired pUC origin for high-copy replication and maintenance of the plasmid in E. coli Kanamycin resistance gene for selection of the plasmid in E. coli
For a map and a description of the features of each donor vector, see the Appendix, pages 41-44.
Resuspending the Donor Vectors
All donor vectors are supplied as 6 g of supercoiled plasmid, lyophilized in TE Buffer, pH 8.0. To use, resuspend the pDONRTM plasmid DNA in 40 l of sterile water to a final concentration of 150 ng/l. To propagate donor vectors, see page 10.
BP ClonaseTM II Enzyme Mix
BP ClonaseTM II enzyme mix is supplied with the kit to catalyze the BP recombination reaction. The BP ClonaseTM II enzyme mix combines the proprietary enzyme formulation and 5X BP ClonaseTM Reaction Buffer previously supplied as separate components in BP ClonaseTM enzyme mix (Catalog no. 11789-019) into an optimized single-tube format to allow easier set-up of the BP recombination reaction. Use the protocol provided on page 26 to perform the BP recombination reaction using BP ClonaseTM II enzyme mix.
Note: You may perform the BP recombination reaction using BP ClonaseTM enzyme mix (not supplied), if desired. To use BP ClonaseTM enzyme mix, follow the protocol provided with the product. Do not use the protocol for BP ClonaseTM II enzyme mix provided on page 26 as reaction conditions differ.
continued on next page
20
Creating Entry Clones Using the BP Recombination Reaction, continued
Recombination Region of the attL4 and attR1Flanked Entry Clone
The recombination region of the entry clone resulting from pDONRTMP4-P1R x attB4-5 element-attB1 is shown below. Features of the Recombination Region: * Shaded regions correspond to those DNA sequences transferred from the attB PCR product into the pDONRTMP4-P1R vector by recombination. Non-shaded regions are derived from the pDONRTMP4-P1R vector. Bases 674 and 2830 of the pDONRTMP4-P1R sequence are marked.
M13 Forward (-20) priming site
*
531 GACGTTGTAA AACGACGGCC AGTCTTAAGC TCGGGCCCGC GTTAACGCTA CCATGGAGCT
591 CCAAATAATG ATTTTATTTT GACTGATAGT GACCTGTTCG TTGCAACAAA TTGATAAGCA GGTTTATTAC TAAAATAAAA CTGACTATCA CTGGACAAGC AACGTTGTTT AACTATTCGT
674 attL4
651 ATGCTTTTTT ATAATGCCA ACT TTG TAT AGA AAA GTT GNN --- --- --- NCA 5 --- --TACGAAAAAA TATTACGGT TGA AAC ATA TCT TTT CAA CNN ---Element NGT
2830
2825 AGT TTG TAC AAA AAA GTT GAACGAGAAA CGTAAAATGA TATAAATATC AATATATTAA TCA AAC ATG TTT TTT CAA CTTGCTCTTT GCATTTTACT ATATTTATAG TTATATAATT
attR1
2883 ATTAGATTTT GCATAAAAAA CAGACTACAT AATACTGTAA AACACAACAT ATGCAGTCAC TAATCTAAAA CGTATTTTTT GTCTGATGTA TTATGACATT TTGTGTTGTA TACGTCAGTG
2943 TATGAATCAA CTACTTAGAT GGTATTAGTG ACCTGTAGAA TTCGAGCTCT AGAGCTGCAG ATACTTAGTT GATGAATCTA CCATAATCAC TGGACATCTT
M13 Reverse priming site
3003 GGCGGCCGCG ATATCCCCTA TAGTGAGTCG TATTACATGG TCATAGCTGT TTCCTGGCAG
continued on next page
21
Creating Entry Clones Using the BP Recombination Reaction, continued
Recombination Region of the attL1 and attL2Flanked Entry Clone
The recombination region of the entry clone resulting from pDONRTM221 x attB1gene of interest-attB2 is shown below. Features of the Recombination Region: * Shaded regions correspond to those DNA sequences transferred from the attB PCR product into the pDONRTM221 vector by recombination. Non-shaded regions are derived from the pDONRTM221 vector. Bases 651 and 2897 of the pDONRTM221 sequence are marked.
M13 Forward (-20) priming site
*
531
GACGTTGTAA AACGACGGCC AGTCTTAAGC TCGGGCCCCA AATAATGATT TTATTTTGAC AGCCCGGGGT TTATTACTAA AATAAAACTG
591
TGATAGTGAC CTGTTCGTTG CAACACATTG ATGAGCAATG CTTTTTTATA ATG CCA ACT ACTATCACTG GACAAGCAAC GTTGTGTAAC TACTCGTTAC GAAAAAATAT TAC GGT TGA
651 attL1
2897
650
TTG TAC AAA AAA GCA GGC TNN --- --- --- NAC CCA GCT TTC TTG TAC AAA AAC ATG TTT TTT CGT CCG ANN --- Gene --- NTG GGT CGA AAG AAC ATG TTT ---
2907
GTT GGC ATT ATAAGAAAGC ATTGCTTATC AATTTGTTGC AACGAACAGG TCACTATCAG CAA CCG TAA TATTCTTTCG TAACGAATAG TTAAACAACG TTGCTTGTCC AGTGATAGTC
attL2
2966
TCAAAATAAA ATCATTATTT GCCATCCAGC TGATATCCCC TATAGTGAGT CGTATTACAT AGTTTTATTT TAGTAATAAA CGGTAGGTCG
M13 Reverse priming site
3026
GGTCATAGCT GTTTCCTGGC AGCTCTGGCC CGTGTCTCAA AATCTCTGAT GTTACATTGC
continued on next page
22
Creating Entry Clones Using the BP Recombination Reaction, continued
Recombination Region of the attR2 and attL3Flanked Entry Clone
The recombination region of the entry clone resulting from pDONRTMP2R-P3 x attB2-3 element-attB3 is shown below. Features of the Recombination Region: * Shaded regions correspond to those DNA sequences transferred from the attB PCR product into the pDONRTMP2R-P3 vector by recombination. Non-shaded regions are derived from the pDONRTMP2R-P3 vector. Bases 733 and 2889 of the pDONRTMP2R-P3 sequence are marked.
M13 Forward (-20) priming site
*
531 GACGTTGTAA AACGACGGCC AGTCTTAAGC TCGGGCCCTG CAGCTCTAGA GCTCGAATTC
591 TACAGGTCAC TAATACCATC TAAGTAGTTG ATTCATAGTG ACTGCATATG TTGTGTTTTA ATGTCCAGTG ATTATGGTAG ATTCATCAAC TAAGTATCAC TGACGTATAC AACACAAAAT
attR2
651 CAGTATTATG TAGTCTGTTT TTTATGCAAA ATCTAATTTA ATATATTGAT ATTTATATCA GTCATAATAC ATCAGACAAA AAATACGTTT TAGATTAAAT TATATAACTA TAAATATAGT
733
711 TTTTACGTTT CTCGTTCA ACT TTC TTG TAC AAA GTG GNN --- --- --- NCA 3 --- --AAAATGCAAA GAGCAAGT TGA AAG AAC ATG TTT CAC CNN ---Element NGT
2889
2884 ACT TTA TTA TAC AAA GTT GGCATTATA AAAAAGCATT GCTTATCAAT TTGTTGCAAC TGA AAT AAT ATG TTT CAA CCGTAATAT TTTTTCGTAA CGAATAGTTA AACAACGTTG
attL3
2941 GAACAGGTCA CTATCAGTCA AAATAAAATC ATTATTTGGA GCTCCATGGT AGCGTTAACG CTTGTCCAGT GATAGTCAGT TTTATTTTAG TAATAAACCT
M13 Reverse priming site
3001 CGGCCGCGAT ATCCCCTATA GTGAGTCGTA TTACATGGTC ATAGCTGTTT CCTGGCAGCT
23
Performing the BP Recombination Reaction
Introduction
General guidelines and instructions are provided below and in the next section to perform a BP recombination reaction using the appropriate attB PCR product and donor vector, and to transform the reaction mixture into a suitable E. coli host to select for entry clones. We recommend including a positive control and a negative control (no BP ClonaseTM II) to help you evaluate your results.
Positive Control
pMS/GW is included with the MultiSite Gateway(R) Three-Fragment Vector Construction Kit for use as a positive control for each BP reaction, and contains multiple DNA fragments that have been joined using MultiSite Gateway(R) Technology (see the Appendix, page 47 for a map and more information). For an alternate positive control to use when creating an attL1 and attL2-flanked entry clone, see below. The pMS/GW plasmid is supplied as 10 g of supercoiled plasmid, lyophilized in TE Buffer, pH 8.0. To use, resuspend the pMS/GW DNA in 10 l of sterile water to a final concentration of 1 g/l. To propagate the plasmid, see page 10.
Linearizing the Positive Control
You will need to linearize the pMS/GW plasmid before it may be used as a control for each BP reaction. We recommend linearizing the vector by restriction digest using Aat II (New England Biolabs, Catalog no. R0117S). 1. 2. 3. Digest 5 g of pMS/GW plasmid in a 50 l reaction using Aat II. Follow the manufacturer's instructions. Incubate the reaction at 70C for 1 hour to inactivate the Aat II. Proceed to Setting Up the BP Reaction, page 26. Note that the concentration of the digested DNA is 100 ng/l.
Alternate Positive Control
When creating attL1 and attL2-flanked entry clones, you may use the pEXP7-tet supplied with the kit as a positive control in a BP reaction with pDONRTM221. pEXP7-tet is an approximately 1.4 kb linear fragment and contains attB1 and attB2 sites flanking the tetracycline resistance gene and its promoter (Tcr).
Determining How Much attB PCR Product and Donor Vector to Use in the Reaction
For optimal efficiency, we recommend using the following amounts of attB PCR product and donor vector in a 10 l BP recombination reaction: * * An equimolar amount of attB PCR product and the donor vector 50 femtomoles (fmoles) each of attB PCR product and donor vector is preferred, but the amount of attB PCR product used may range from 2050 fmoles
Note: 50 fmoles of donor vector (pDONRTMP4-P1R, pDONRTM221, or pDONRTMP2R-P3) is approximately 150 ng
*
For large PCR products (>4 kb), use at least 50 fmoles of attB PCR product, but no more than 250 ng
For a formula to convert fmoles of DNA to nanograms (ng) and an example, see the next page. continued on next page 24
Performing the BP Recombination Reaction, continued
* * Do not use more than 250 ng of donor vector in a 10 l BP reaction as this will affect the efficiency of the reaction. Do not exceed more than 500 ng of total DNA (donor vector plus attB PCR product) in a 10 l BP reaction as excess DNA will inhibit the reaction.
Converting Femto- Use the following formula to convert femtomoles (fmoles) of DNA to nanograms (ng) of DNA: moles (fmoles) to Nanograms (ng) 660 fg 1 ng
ng = (x fmoles)(N)(
)( ) fmoles 10 6 fg
where x is the number of fmoles and N is the size of the DNA in bp. For an example, see below.
Example of fmoles In this example, you need to use 50 fmoles of an attB PCR product in the BP reaction. The attB PCR product is 2.5 kb in size. Calculate the amount of attB PCR to ng Conversion
product required for the reaction (in ng) by using the equation above:
(50 fmoles)(2500 bp)(
660 fg 1 ng )( ) = 82.5 ng of PCR product required fmoles 10 6 fg
Materials Needed
You should have the following materials on hand before beginning. Supplied with the kit: * * * * * * pDONRTM vectors (i.e. pDONRTMP4-P1R, pDONRTM221, and pDONRTMP2R-P3; resuspend each vector to 150 ng/l with water) BP ClonaseTM II enzyme mix (keep at -20C until immediately before use) 2 g/l Proteinase K solution (thaw and keep on ice until use) pMS/GW control plasmid (linearize before use; 100 ng/l) pEXP7-tet positive control (50 ng/l; optional) attB PCR products (i.e. attB4-PCR product-attB1, attB1-PCR product-attB2, or attB2-PCR product-attB3; see the previous page and above to determine the amount of DNA to use) TE Buffer, pH 8.0 (10 mM Tris-HCl, pH 8.0, 1 mM EDTA) continued on next page
Supplied by the user:
*
25
Performing the BP Recombination Reaction, continued
Setting Up the BP Reaction
1. For each BP recombination reaction between an appropriate attB PCR product and donor vector, add the following components to 1.5 ml microcentrifuge tubes at room temperature and mix.
Note: If you are using pEXP7-tet as a positive control, use 100 ng (2 l) in place of the pMS/GW DNA.
Components attB PCR product (20-50 fmoles) pDONRTM vector (150 ng/l) pMS/GW control plasmid (100 ng/l) TE Buffer, pH 8.0 2. 3. 4.
Sample 1-7 l 1 l -to 8 l
Negative Control 1-7 l 1 l -to 10 l --
Positive Control 1 l 2 l 5 l
Remove the BP ClonaseTM II enzyme mix from -20C and thaw on ice (~ 2 minutes). Vortex the BP ClonaseTM II enzyme mix briefly twice (2 seconds each time). To each sample above, add 2 l of BP ClonaseTM II enzyme mix. Mix well by vortexing briefly twice (2 seconds each time).
Reminder: Return BP ClonaseTM II enzyme mix to -20C immediately after use.
5.
Incubate reactions at 25C for 1 hour.
Note: A 1 hour incubation generally yields a sufficient number of entry clones. Depending on your needs, the length of the recombination reaction can be extended up to 18 hours. An overnight incubation typically yields 5-10 times more colonies than a 1 hour incubation. For large PCR products (5 kb), longer incubations (i.e. overnight incubation) will increase the yield of colonies and are recommended.
6. 7.
Add 1 l of the Proteinase K solution to each reaction. Incubate for 10 minutes at 37C. Proceed to Transforming One Shot(R) TOP10 Competent Cells, next page.
Note: You may store the BP reaction at -20C for up to 1 week before transformation, if desired.
26
Transforming One Shot(R) TOP10 Competent Cells
Introduction
Use the guidelines and procedures provided in this section to transform competent E. coli with the BP recombination reaction or the MultiSite Gateway(R) LR recombination reaction to select for entry clones or expression clones, respectively. One Shot(R) TOP10 chemically competent E. coli (Box 4) are included with the kit for use in transformation, however, you may also transform electrocompetent cells. Instructions to transform chemically competent or electrocompetent E. coli are provided in this section.
You may use any recA, endA E. coli strain including TOP10 (supplied with the kit), DH5TM, DH10BTMor equivalent for transformation. Other strains are suitable. Do not use E. coli strains that contain the F episome (e.g. TOP10F) for transformation. These strains contain the ccdA gene and will prevent negative selection with the ccdB gene. For your convenience, TOP10, DH5TM, and DH10BTM E. coli are available separately from Invitrogen as chemically competent or electrocompetent cells (see table below). Item Library Efficiency DH5
(R) (R) TM
Quantity 5 x 200 l 20 x 50 l 20 x 50 l 20 x 50 l 5 x 100 l
Catalog No. 18263-012 C4040-03 12331-013 C4040-52 18290-015
One Shot TOP10 Chemically Competent E. coli One Shot(R) Max Efficiency(R) DH10BTM T1 Phage Resistant Chemically Competent E. coli One Shot(R) TOP10 Electrocomp E. coli ElectroMax DH10B
TM TM
Materials Needed
You should have the following materials on hand before beginning. Supplied with the kit: * * * * One Shot(R) TOP10 chemically competent E. coli (thaw on ice 1 vial of One Shot(R) TOP10 cells for each transformation) S.O.C. medium (warm to room temperature) Positive control (e.g. pUC19; use as a control for transformation if desired) BP recombination reaction (from Setting Up the BP Reaction, Step 7, previous page) or MultiSite Gateway(R) LR recombination reaction (from Setting Up the MultiSite Gateway(R) LR Reaction, Step 7, page 36) LB plates containing 50 g/ml kanamycin (for the BP reaction) or 50100 g/ml ampicillin (for the MultiSite Gateway(R) LR reaction). Prepare two plates for each transformation; warm at 37C for 30 minutes. 42C water bath (for chemical transformation) 37C shaking and non-shaking incubator continued on next page 27
Supplied by the user:
*
* *
Transforming One Shot(R) TOP10 Competent Cells, continued
One Shot(R) TOP10 Chemical Transformation Protocol
1. Into a vial of One Shot(R) TOP10 chemically competent E. coli, add the following and mix gently. Do not mix by pipetting up and down. * * Add 1 l of the BP recombination reaction (from Setting Up the BP Reaction, Step 7, page 26) or Add 2 l of the MultiSite Gateway(R) LR recombination reaction (from Setting Up the MultiSite Gateway(R) LR Reaction, Step 7, page 36). Note: You may transform up to 5 l of the reaction, if desired.
Reminder: If you are including the transformation control, add 1 l (10 pg) of pUC19.
2. 3. 4. 5. 6. 7.
Incubate on ice for 5 to 30 minutes. Heat-shock the cells for 30 seconds at 42C without shaking. Immediately transfer the tubes to ice. Add 250 l of room temperature S.O.C. medium. Cap the tube tightly and shake the tube horizontally (200 rpm) at 37C for 1 hour. Spread the following amount from each transformation on a prewarmed selective plate and incubate overnight at 37C. We generally plate 2 different volumes to ensure that at least 1 plate has well-spaced colonies. * * BP recombination reaction: spread 20 l and 100 l MultiSite Gateway(R) LR recombination reaction: spread 50 l and 100 l
What You Should See
*
BP reaction: An efficient BP recombination reaction may produce hundreds of colonies (greater than 1,500 colonies if the entire reaction is transformed and plated). MultiSite Gateway(R) LR reaction: An efficient MultiSite Gateway(R) LR recombination reaction may produce approximately 100 colonies (approximately 2,000 to 8,000 if the entire reaction is transformed and plated). continued on next page
*
28
Transforming One Shot(R) TOP10 Competent Cells, continued
Transformation by Electroporation
Use only electrocompetent cells for electroporation to avoid arcing. Do not use the One Shot(R) TOP10 chemically competent cells for electroporation. 1. Into a 0.1 cuvette containing 50 l of electrocompetent E. coli, add the following and mix gently. Do not mix by pipetting up and down. Avoid formation of bubbles. * * 2. 1 l of the BP recombination reaction (from Setting Up the BP Reaction, Step 7, page 26) or 2 l of the MultiSite Gateway(R) LR recombination reaction (from Setting Up the MultiSite Gateway(R) LR Reaction, Step 7, page 36).
Electroporate your samples using an electroporator and the manufacturer's suggested protocol.
Note: If you have problems with arcing, see below.
3. 4. 5.
Immediately add 450 l of room temperature S.O.C. medium. Transfer the solution to a 15 ml snap-cap tube (i.e. Falcon) and shake for at least 1 hour at 37C to allow expression of the antibiotic resistance marker. Spread 50-100 l from each transformation on a prewarmed selective plate and incubate overnight at 37C. We recommend plating 2 different volumes to ensure that at least 1 plate has well-spaced colonies. An efficient recombination reaction may produce several hundred colonies.
6.
RECOM
M EN D
To prevent arcing of your samples during electroporation, the volume of cells should be between 50 and 80 l (0.1 cm cuvettes) or 100 to 200 l (0.2 cm cuvettes). If you experience arcing during transformation, try one of the following: * * * Reduce the voltage normally used to charge your electroporator by 10% Reduce the pulse length by reducing the load resistance to 100 ohms Dilute the BP reaction 5-10 fold with sterile water, then transform 1 l into cells
ION AT
29
Sequencing Entry Clones
Introduction
You may sequence entry clones generated by BP recombination using dye-labeled terminator chemistries including DYEnamicTM energy transfer or BigDyeTM reaction chemistries.
Sequencing Primers
To sequence entry clones derived from BP recombination with pDONRTMP4-P1R, pDONRTM221, and pDONRTMP2R-P3, we recommend using the following sequencing primers: Forward primer Reverse primer M13 Forward (-20): 5-GTAAAACGACGGCCAG-3 M13 Reverse: 5-CAGGAAACAGCTATGAC-3
See the diagrams on page 21-23 for the location of the M13 forward (-20) and M13 reverse primer binding sites in each entry clone. The M13 Forward (-20) and M13 Reverse Primers (Catalog nos. N520-02 and N530-02, respectively) are available separately from Invitrogen. For more information, see our Web site (www.invitrogen.com) or call Technical Service (see page 48).
Sequencing Using BigDyeTM Chemistry
To sequence entry clones using the BigDyeTM chemistry, we recommend the following: * * * Use at least 500 ng of DNA Use 5-50 pmoles of primers Use 1/4 reaction and the PCR conditions listed below
PCR Conditions
Use the following PCR conditions for sequencing using BigDyeTM chemistry. These conditions are suitable for most inserts, including small inserts. Step Initial Denaturation Denaturation Annealing Extension Time 5 minutes 10-30 seconds 5-15 seconds 4 minutes Temperature 95C 96C 50C 60C 30X Cycles 1X
BigDyeTM is a registered trademark of Applied Biosystems
30
Creating Expression Clones Using the MultiSite Gateway(R) LR Recombination Reaction
Introduction
After you have generated entry clones containing your 5 element, gene of interest, and 3' element, you will perform the MultiSite Gateway(R) LR recombination reaction to simultaneously transfer the three DNA fragments into the pDESTTMR4-R3 destination vector to create an attB-containing expression clone with the following structure: attB4-5 element-attB1-gene of interest-attB2-3 element-attB3 To ensure that you obtain the best results, we suggest reading this section and the next section entitled Performing the MultiSite Gateway(R) LR Recombination Reaction (pages 34-36) before beginning.
Experimental Outline
To generate an expression clone, you will: 1. 2. 3. Perform a MultiSite Gateway(R) LR recombination reaction using the appropriate entry clones and pDESTTMR4-R3 (see below). Transform the reaction mixture into a suitable E. coli host (see page 27). Select for MultiSite Gateway(R) expression clones (see page 33 for a diagram of the recombination region).
Substrates for the MultiSite Gateway(R) LR Recombination Reaction
To perform a three-fragment MultiSite Gateway(R) LR recombination reaction, you must have the substrates listed below. * * * * * attL4 and attR1-containing entry clone attL1 and attL2-containing entry clone attR2 and attL3-containing entry clone pDESTTMR4-R3 destination vector (see the next page for more information) You cannot successfully create a three-fragment expression clone using the MultiSite Gateway(R) LR recombination reaction if you have any combination of att-flanked entry clones other than the ones listed above. You must use the pDESTTMR4-R3 destination vector for the three-fragment MultiSite Gateway(R) LR recombination reaction. Other Gateway(R) destination vectors cannot be used.
Keep in mind the following:
*
Important
For optimal results, we recommend performing the MultiSite Gateway(R) LR recombination reaction using: * * Supercoiled entry clones Supercoiled pDESTTMR4-R3 continued on next page
31
Creating Expression Clones Using the MultiSite Gateway(R) LR Recombination Reaction, continued
pDESTTMR4-R3 Vector
The pDESTTMR4-R3 vector is supplied with the kit for use in the MultiSite Gateway(R) LR recombination reaction to generate an expression clone containing your three DNA fragments of choice. The pDESTTMR4-R3 plasmid contains the following elements: * * attR4 and attR3 sites for recombinational cloning of three DNA fragments from the appropriate Gateway(R) entry clones M13 forward (-20) and M13 reverse primer binding sites to facilitate sequencing of the expression clone, if desired
Note: When sequencing your expression clone, you will use the M13 reverse primer to sequence the sense strand and the M13 forward (-20) primer to sequence the anti-sense strand. Refer to the diagram on page 33 for the location of the priming sites.
* *
pUC origin for high-copy replication and maintenance of the plasmid in E. coli Ampicillin resistance gene for selection of the plasmid in E. coli
Important: Note that all other elements required to express your gene of interest in the system of choice must be supplied by the entry clones.
Resuspending the pDESTTMR4-R3 Vector Determining How Much DNA to Use in the Reaction
pDESTTMR4-R3 is supplied as 6 g of plasmid, lyophilized in TE, pH 8.0. To use, resuspend the destination plasmid in 100 l of sterile water to a final concentration of 60 ng/l. To propagate the vector, see page 10.
For optimal efficiency, we recommend using the following amounts of plasmid DNA (i.e. entry clones and destination vector) in a 20 l MultiSite Gateway(R) LR recombination reaction: * * An equimolar amount of each plasmid 20-25 fmoles of each entry clone and pDESTTMR4-R3 is recommended. Do not use more than 30 fmoles of each plasmid.
Note: 20 fmoles of pDESTTMR4-R3 is approximately 60 ng
For a formula to convert fmoles of DNA to nanograms (ng) and an example, see page 25.
* *
Do not use more than 120 fmoles of total plasmid DNA in a 20 l MultiSite Gateway(R) LR reaction as this will affect the efficiency of the reaction. Do not exceed more than 1 g of total DNA (i.e. 250 ng of each entry clone plus destination vector) in a 20 l MultiSite Gateway(R) LR reaction as excess DNA will inhibit the reaction. If you need to use more than 1 g of total DNA, scale up the volume of the MultiSite Gateway(R) LR reaction. continued on next page
32
Creating Expression Clones Using the MultiSite Gateway(R) LR Recombination Reaction, continued
Recombination Region of the Expression Clone
The recombination region of the expression clone resulting from pDESTTMR4-R3 x attL4-5 entry clone-attR1 x attL1-entry clone-attL2 x attR2-3 entry clone-attL3 is shown below. Features of the Recombination Region: * Shaded regions correspond to those DNA sequences transferred from the three entry clones into the pDESTTMR4-R3 vector by recombination. Note that the sequences comprising the attB1 and attB2 sites are entirely supplied by the entry clones. Non-shaded regions are derived from the pDESTTMR4-R3 vector. Bases 43 and 1867 of the pDESTTMR4-R3 sequence are indicated.
43
*
M13 Reverse priming site
1 CAGGAAACAG CTATGACCAT GATTACGCCA AGCTATCAACT TTG TAT AGA AAA GTT GTCCTTTGTC GATACTGGTA CTAATGCGGT TCGATAGTTGA AAC ATA TCT TTT CAA
attB4
57 GNN --- --- --- NCA AGT TTG TAC AAA AAA GCA GGC TNN --- --- --- NAC 5 Element CNN --- --- --- NGT TCA AAC ATG TTT TTT CGT CCG ANN --- Gene --- NTG --attB1
1727
CCA GCT TTC TTG TAC AAA GTG GNN --- --- --- NCA ACT TTA TTA TAC ATA 3 --- --GGT CGA AAG AAC ATG TTT CAC CNN --- Element NGT TGA AAT AAT ATG TAT
attB2
M13 Forward (-20) priming site
attB3
1877 GTTGATAATT CACTGGCCGT CGTTTTACAA CGTCGTGACT GGGAAAACCC TGGCGTTACC CAACTATTAA GTGACCGGCA GCAAAATGTT GCAGCACTGA CCCTTTTGGG ACCGCAATGG
33
Performing the MultiSite Gateway(R) LR Recombination Reaction
Introduction
Guidelines and instructions are provided in this section to: * * * Perform a MultiSite Gateway(R) LR recombination reaction between suitable entry clones and pDESTTMR4-R3 using LR ClonaseTM Plus enzyme mix. Transform the reaction mixture into a suitable E. coli host (see below) Select for an expression clone
We recommend including a positive control (see below) and a negative control (no LR ClonaseTM Plus) in your experiment to help you evaluate your results.
E. coli Host
We recommend using the One Shot(R) TOP10 Chemically Competent E. coli supplied with the kit for transformation. If you wish to use another E. coli strain, note that any recA, endA E. coli strain is suitable. Do not transform the LR reaction mixture into E. coli strains that contain the F episome (e.g. TOP10F). These strains contain the ccdA gene and will prevent negative selection with the ccdB gene.
Note: To use the One Shot(R) TOP10 chemically competent cells for transformation, see the section entitled Transforming One Shot(R) TOP10 Competent Cells, pages 27-29.
Positive Control
If you used the pMS/GW plasmid as a control for each BP recombination reaction, you may use the resulting three entry clones as controls in a MultiSite Gateway(R) LR recombination reaction with pDESTTMR4-R3.
Preparing Purified Plasmid DNA
You will need to have purified plasmid DNA of each entry clone to perform the MultiSite Gateway(R) LR recombination reaction. You may use any method of choice to isolate purified plasmid DNA. We recommend using the S.N.A.P.TM MidiPrep Kit (Catalog no. K1910-01) or the PureLinkTM HQ Mini Plasmid Purification Kit (Catalog no. K2100-01) available from Invitrogen.
Important
You must use LR ClonaseTM Plus enzyme mix to catalyze the MultiSite Gateway(R) LR recombination reaction. Note that the LR ClonaseTM II enzyme mix (Catalog no. 11791-020) used for standard Gateway(R) LR recombination reactions cannot be used for MultiSite Gateway(R) LR recombination reactions. LR ClonaseTM Plus enzyme mix is supplied with the kit, but is also available separately from Invitrogen (see page ix for ordering information). continued on next page
34
Performing the MultiSite Gateway(R) LR Recombination Reaction, continued
Materials Needed
You should have the following materials on hand before beginning. Supplied with the kit: * * * * * * * pDESTTMR4-R3 (60 ng/l in TE, pH 8.0) LR ClonaseTM Plus enzyme mix (Box 3, keep at -80C until immediately before use) 5X LR ClonaseTM Plus Reaction Buffer (thaw and keep on ice before use) 2 g/l Proteinase K solution Purified plasmid DNA of your attL4 and attR1-flanked entry clone (supercoiled, 20-25 fmoles) Purified plasmid DNA of your attL1 and attL2-flanked entry clone (supercoiled, 20-25 fmoles) Purified plasmid DNA of your attR2 and attL3-flanked entry clone (supercoiled, 20-25 fmoles)
Important: Remember that you will need to add plasmid DNA from three entry clones to the MultiSite Gateway(R) LR reaction. Make sure that the plasmid DNA for each entry clone is sufficiently concentrated such that the total amount of entry clone plasmid DNA added to a 20 l MultiSite Gateway(R) LR reaction does not exceed 11 l.
Supplied by the user:
* * * *
TE Buffer, pH 8.0 (10 mM Tris-HCl, pH 8.0, 1 mM EDTA) Appropriate competent E. coli host (e.g. One Shot(R) TOP10) and growth media for expression S.O.C. Medium LB agar plates containing 50-100 g/ml ampicillin continued on next page
35
Performing the MultiSite Gateway(R) LR Recombination Reaction, continued
1. Add the following components to 1.5 ml microcentrifuge tubes at room Setting Up the (R) temperature and mix. MultiSite Gateway LR Reaction Component attL4 and attR1 entry clone (20-25 fmoles) attL1 and attL2 entry clone (20-25 fmoles) attR2 and attL3 entry clone (20-25 fmoles) pDESTTMR4-R3 vector (60 ng/reaction) 5X LR Clonase Plus Reaction Buffer TE Buffer, pH 8.0 2. 3. 4.
TM
Sample 3-11 l
Negative Control --
1 l 4 l to 16 l
1 l 4 l 11 l
Remove the LR ClonaseTM Plus enzyme mix from -80C and thaw on ice (~ 2 minutes). Vortex the LR ClonaseTM Plus enzyme mix briefly twice (2 seconds each time). To each sample above, add 4 l of LR ClonaseTM Plus enzyme mix. Mix well by vortexing briefly twice (2 seconds each time).
Reminder: Return LR ClonaseTM Plus enzyme mix to -80C immediately after use.
5. 6. 7.
Incubate reactions at 25C for 16 hours or overnight. Add 2 l of the Proteinase K solution to each reaction. Incubate for 10 minutes at 37C. Proceed to transform a suitable E. coli host and select for expression clones. If you are transforming One Shot(R) TOP10 chemically competent E. coli, follow the protocol on page 28.
Note: You may store the MultiSite Gateway(R) LR reaction at -20C for up to 1 week before transformation, if desired.
What You Should See
If you use E. coli cells with a transformation efficiency of 1 x 109 cfu/g, the MultiSite Gateway(R) LR reaction should give approximately 2,000 to 8,000 colonies if the entire reaction is transformed and plated. Once you have obtained an expression clone, proceed to express your recombinant protein in the appropriate system.
36
Troubleshooting
MultiSite Gateway(R) The table below lists some potential problems and possible solutions that may LR & BP Reactions help you troubleshoot the BP or MultiSite Gateway(R) LR recombination reactions.
Problem Few or no colonies obtained from sample reaction and the transformation control gave colonies Reason Incorrect antibiotic used to select for transformants Recombination reactions were not treated with proteinase K Used incorrect att sites for the reaction Solution Check the antibiotic resistance marker and use the correct antibiotic to select for entry clones or expression clones. Treat reactions with proteinase K before transformation. * Use the appropriate entry clones and pDESTTMR4-R3 for the MultiSite Gateway(R) LR reaction (see page 11 for details about the types of entry clones required). Use the correct attB PCR product and donor vector (attP) for the BP reaction (see page 19 for details). Test another aliquot of the ClonaseTM (Plus) enzyme mix. Store the LR ClonaseTM Plus at -80C and the BP ClonaseTM II at -20C. Do not freeze/thaw the ClonaseTM (Plus) enzyme mix more than 10 times. Use the recommended amount of ClonaseTM (Plus) enzyme mix (see page 26 or 36 as appropriate). Use the LR ClonaseTM Plus enzyme mix for the MultiSite Gateway(R) LR reaction. Do not use the LR ClonaseTM enzyme mix. Use the BP ClonaseTM II enzyme mix for the BP reaction.
*
ClonaseTM (Plus) enzyme mix is inactive or didn't use suggested amount of ClonaseTM (Plus) enzyme mix
* * *
*
Used incorrect ClonaseTM enzyme mix
*
* Too much attB PCR product was used in a BP reaction
Reduce the amount of attB PCR product used. Use an equimolar ratio of attB PCR product and donor vector (i.e. ~50 fmoles each). Incubate the BP reaction overnight. Use an equimolar amount of each entry clone and destination vector. Do not exceed 120 fmoles or 1 g of total DNA in the reaction. continued on next page 37
Long attB PCR product or linear attB expression clone (5 kb) Too much DNA was used in a MultiSite Gateway(R) LR reaction
Troubleshooting
MultiSite Gateway(R) LR and BP Reactions, continued
Problem Few or no colonies obtained from sample reaction and the transformation control gave colonies, continued Reason MultiSite Gateway LR reaction not incubated for sufficient time Insufficient amount of E. coli transformed or plated
(R)
Solution Incubate the MultiSite Gateway(R) LR reaction at 25C for 16 hours or overnight. MultiSite Gateway(R) LR reaction: Transform 2 to 5 l of the reaction; plate 50 l or 100 l. BP reaction: Transform 1 l of the reaction; plate 20 l and 100 l.
MultiSite Gateway(R) LR Reaction: High background in the absence of the entry clones
MultiSite Gateway(R) LR reaction transformed into an E. coli strain containing the F episome and the ccdA gene Deletions (full or partial) of the ccdB gene from the destination vector
Use an E. coli strain that does not contain the F episome for transformation (e.g. TOP10, DH5TM). * To maintain the integrity of the vector, propagate in media containing 50-100 g/ml ampicillin and 15-30 g/ml chloramphenicol. Prepare plasmid DNA from one or more colonies and verify the integrity of the vector before use. Test for plasmid contamination by transforming E. coli with aliquots of each of the separate solutions used in the MultiSite Gateway(R) LR reaction. Test for bacterial contamination by plating an aliquot of each solution directly onto LB plates containing ampicillin.
*
Contamination of solution(s) with another plasmid carrying the same antibiotic resistance, or by bacteria carrying a resistance plasmid
*
*
Few or no colonies obtained from the transformation control
Competent cells stored incorrectly Transformation performed incorrectly
Store competent cells at -80C. If you are using One Shot(R) TOP10 E. coli, follow the protocol on page 28 to transform cells. If you are using another E. coli strain, follow the manufacturer's instructions.
Insufficient amount of E. coli plated
Increase the amount of E. coli plated.
continued on next page
38
Troubleshooting
MultiSite Gateway(R) LR and BP Reactions, continued
Problem Two distinct types of colonies (large and small) appear Reason
TM
Solution
BP reaction: The pDONR vector Obtain a new pDONRTM vector. contains deletions or point mutations in the ccdB gene
Note: The negative control will give a similar number of colonies
Loss of plasmid during culture (generally those containing large genes or toxic genes)
* *
Incubate selective plates at 30C instead of 37C. Confirm whether a deletion has occurred by analyzing the DNA derived from the colonies. Use Stbl2TM E. coli (Invitrogen, Catalog no. 10268-019) to help stabilize plasmids containing large genes during propagation (Trinh et al., 1994).
*
attB PCR Cloning
The table below lists some potential problems and possible solutions that may help you troubleshoot the BP recombination reaction when using an attB PCR product as a substrate. These potential problems are in addition to those encountered in the general BP reaction (see page 37). Reason attB PCR product not diluted with TE Centrifugation step too short or centrifugation speed too low Lost PEG pellet Solution Dilute with 150 l of 1X TE, pH 8.0 before adding the PEG/MgCl2 solution. Increase time and speed of the centrifugation step to 30 minutes and 15,000 x g. * When removing the tube from the microcentrifuge, keep track of the orientation of the outer edge of the tube where the pellet is located. When removing the supernatant from the tube, take care not to disturb the pellet. continued on next page
Problem Low yield of attB PCR product obtained after PEG purification
*
39
Troubleshooting
attB PCR Cloning, continued
Problem Few or no colonies obtained from a BP reaction with attB PCR product and both attB positive control and transformation control gave expected number of colonies Reason attB PCR primers incorrectly designed Solution Make sure that each attB PCR primer includes four 5 terminal Gs and the 22 or 25 bp attB site as specified on page 15. Use HPLC or PAGE-purified oligonucleotides to generate your attB PCR product. Gel purify your attB PCR product to remove attB primers and attB primerdimers. * Increase the amount of attB PCR product to 20-50 fmoles per 10 l reaction.
Note: Do not exceed 250 ng DNA per 10 l reaction.
attB PCR primers contaminated with incomplete sequences attB PCR product not purified sufficiently For large PCR products (>5 kb), too few attB PCR molecules added to the BP reaction
* Insufficient incubation time Entry clones migrate as 2.2 kb supercoiled plasmids
Incubate the BP reaction overnight.
Increase the incubation time of the BP reaction up to 18 hours. Purify attB PCR product using the PEG/MgCl2 purification protocol on page 18 or gel-purify the attB PCR product. Use a Platinum(R) DNA polymerase with automatic hot-start capability for higher specificity amplification. Redesign attB PCR primers to minimize potential mutual priming sites leading to primer-dimers.
BP reaction may have cloned attB * primer-dimers
*
*
40
Appendix Map of pDONRTMP4-P1R
pDONRTMP4-P1R Map
The map below shows the elements of pDONRTMP4-P1R. The complete sequence of pDONRTMP4-P1R is available from our Web site (www.invitrogen.com) or by contacting Technical Service (see page 48).
M13 Forward M13 Reverse
attP4
ccdB
T1
CmR
attP1R
T
2
ori pUC
pDONR P4-P1R
4777 bp
TM
Comments for pDONRTMP4-P1R 4777 nucleotides
n Ka
am
rrnB T2 transcription termination sequence: bases 268-295 (c) rrnB T1 transcription termination sequence: bases 427-470 (c) M13 Forward (-20) priming site: bases 537-552 attP4 recombination site: bases 593-824 (c) ccdB gene: bases 1181-1486 (c) Chloramphenicol resistance gene: bases 1828-2487 (c) attP1R recombination site: bases 2748-2979 (c) M13 Reverse priming site: bases 3042-3058 Kanamycin resistance gene: bases 3171-3980 pUC origin: bases 4101-4774 (c) = complementary strand
yc
in
Ampicilli
n
41
Map of pDONRTM221
pDONRTM221 Map
The map below shows the elements of pDONRTM221. The complete sequence of pDONRTM221 is available from our Web site (www.invitrogen.com) or by contacting Technical Service (see page 48).
M13 Forward
M13 Reverse
attP1
ccdB
CmR
attP2
T
2
T1
ori pUC
pDONR 221 4762 bp
TM
n Ka
Comments for pDONRTM221 4762 nucleotides rrnB T2 transcription termination sequence (c): bases 268-295 rrnB T1 transcription termination sequence (c): bases 427-470 M13 Forward (-20) priming site: bases 537-552 attP1: bases 570-801 ccdB gene (c): bases 1197-1502 Chloramphenicol resistance gene (c): bases 1847-2506 attP2 (c): bases 2754-2985 M13 Reverse priming site: bases 3027-3043 Kanamycin resistance gene: bases 3156-3965 pUC origin: bases 4086-4759 (c) = complementary strand
am
42
yc
in
Ampicilli
n
Map of pDONRTMP2R-P3
pDONRTMP2R-P3 Map
The map below shows the elements of pDONRTMP2R-P3. The complete sequence of pDONRTMP2R-P3 is available from our Web site (www.invitrogen.com) or by contacting Technical Service (see page 48).
M13 Forward M13 Reverse
attP2R
CmR
T1
ccdB
ATTP3
T
2
ori pUC
pDONR P2R-P3
4773 bp
TM
Comments for pDONRTMP2R-P3 4773 nucleotides
n Ka
am
rrnB T2 transcription termination sequence: bases 268-295 (c) rrnB T1 transcription termination sequence: bases 427-470 (c) M13 Forward (-20) priming site: bases 537-552 attP2R recombination site: bases 591-822 Chloramphenicol resistance gene: bases 1083-1742 ccdB gene: bases 2084-2389 ATTP3 recombination site: bases 2746-2977 M13 Reverse priming site: bases 3038-3054 Kanamycin resistance gene: bases 3167-3976 pUC origin: bases 4097-4770 (c) = complementary strand
yc
in
Ampicilli
n
43
Features of pDONRTM Vectors
Features of the pDONRTM Vectors
pDONRTMP4-P1R (4777 bp), pDONRTM221 (4762 bp), and pDONRTMP2R-P3 (4773 bp) contain the following elements. All features have been functionally tested. Feature rrnB T1 and T2 transcription terminators Benefit Protects the cloned gene from expression by vector-encoded promoters, thereby reducing possible toxicity (Orosz et al., 1991). Allows sequencing in the sense orientation. Bacteriophage -derived DNA recombination sequences that have been optimized to permit recombinational cloning of DNA fragments from specific attB PCR products (Landy, 1989). Permits negative selection of the plasmid.
R
M13 forward (-20) priming site attP4 and attP1R site (pDONR P4-P1R) attP1 and attP2 sites (pDONR 221) attP2R and ATTP3 sites (pDONRTMP2R-P3) ccdB gene Chloramphenicol resistance gene (Cm ) M13 reverse priming site Kanamycin resistance gene pUC origin and replisome assembly site
TM TM
Allows counterselection of the plasmid. Permits sequencing in the anti-sense orientation. Allows selection of the plasmid in E. coli. Permits high-copy replication and maintenance of the plasmid in E. coli.
44
Map and Features of pDESTTMR4-R3
pDESTTMR4-R3 Map
The map below shows the elements of pDESTTMR4-R3. The complete sequence of pDESTTMR4-R3 is available from our Web site (www.invitrogen.com) or by contacting Technical Service (see page 48).
M13 Reverse M13 Forward
attR4
ccdB
CmR
attR3
ri pUC o
pDEST R4-R3
4107 bp
TM
Comments for pDESTTMR4-R3 4107 nucleotides
pi Am
M13 Reverse priming site: bases 1-17 attR4 recombination site: bases 37-161 ccdB gene: bases 201-506 (c) Chloramphenicol resistance gene: bases 848-1507 (c) attR3 recombination site: bases 1616-1740 (c) M13 Forward (-20) priming site: bases 1749-1764 (c) bla promoter: bases 2244-2342 Ampicillin (bla) resistance gene: bases 2343-3203 pUC origin: bases 3348-4021 (c) = complementary strand
continued on next page
in
Ampicilli
n
l ci
45
Map and Features of pDESTTMR4-R3
Features of the pDESTTMR4-R3 Vector
pDESTTMR4-R3 (4107 bp) contains the following elements. All features have been functionally tested. Feature M13 reverse priming site attR4 and attR3 sites Benefit Permits sequencing in the sense orientation. Bacteriophage -derived DNA recombination sequences that have been optimized to permit recombinational cloning of DNA fragments from specific attL-flanked entry clones (Landy, 1989). Permits negative selection of the plasmid.
R
ccdB gene Chloramphenicol resistance gene (Cm ) M13 forward (-20) priming site bla promoter Ampicillin resistance gene (-lactamase) pUC origin and replisome assembly site
Allows counterselection of the plasmid. Allows sequencing in the anti-sense orientation. Permits expression of the ampicillin resistance gene. Allow selection of the plasmid in E. coli. Permits high-copy replication and maintenance of the plasmid in E. coli.
46
Map of pMS/GW
Description
pMS/GW is a 5898 bp control vector, and was generated using the MultiSite Gateway(R) LR recombination reaction between pDESTTMR4-R3 and three entry clones containing the araC gene and araBAD promoter, gus gene, and lacZ fragment, respectively. This expression clone is designed for use as a control for each BP recombination reaction (see page 24 for details).
Map of pMS/GW
The map below shows the elements of pMS/GW. The complete sequence of pMS/GW is available from our Web site (www.invitrogen.com) or by contacting Technical Service (see page 48).
araC
PBAD
M13 Reverse attB4
attB1
gus
attB2
lacZa
attB3 Forward
M13
i pUC or
pMSGW
5898 bp
pi Am
Aat II
Ampicilli
n
Comments for pMSGW 5898 nucleotides M13 Reverse priming site: bases 1-17 attB4 recombination site: bases 37-57 AraC ORF: bases 58-936 (c) Arabinose O2 operator region: bases 966-981 Arabinose O1 operator region: bases 1123-1144 CAP binding site: bases 1165-1178 Arabinose I1 and I2 region: bases 1175-1213 Arabinose minimal promoter: bases 1175-1213 Ribosome binding site: bases 1267-1270 attB1 recombination site: bases 1285-1308 gus gene: bases 1306-3149 attB2 recombination site: bases 3154-3174 lacZa gene: bases 3175-3509 attB3 recombination site: bases 3510-3530 M13 Forward (-20) priming site: bases 3539-3554 Aat II linearization site: base 4002 bla promoter: bases 4034-4132 Ampicillin (bla) resistance gene: bases 4133-4993 pUC origin: bases 5138-5811 (c) = complementary strand
l ci
in
47
Technical Service
World Wide Web
Visit the Invitrogen Web Resource using your World Wide Web browser. At the site, you can: * * * * * * Get the scoop on our hot new products and special product offers View and download vector maps and sequences Download manuals in Adobe(R) Acrobat(R) (PDF) format Explore our catalog with full color graphics Obtain citations for Invitrogen products Request catalog and product literature
Once connected to the Internet, launch your web browser (Netscape 3.0 or newer), then enter the following location (or URL): http://www.invitrogen.com ...and the program will connect directly. Click on underlined text or outlined graphics to explore. Don't forget to put a bookmark at our site for easy reference!
Contact Us
For more information or technical assistance, please call, write, fax, or email. Additional international offices are listed on our web page (www.invitrogen.com). Japanese Headquarters: Invitrogen Japan K.K. Nihonbashi Hama-Cho Park Bldg. 4F 2-35-4, Hama-Cho, Nihonbashi Tel: 81 3 3663 7972 Fax: 81 3 3663 8242 E-mail: jpinfo@invitrogen.com European Headquarters: Invitrogen Ltd Inchinnan Business Park 3 Fountain Drive Paisley PA4 9RF, UK Tel: +44 (0) 141 814 6100 Tech Fax: +44 (0) 141 814 6117 E-mail: eurotech@invitrogen.com
Corporate Headquarters: Invitrogen Corporation 1600 Faraday Avenue Carlsbad, CA 92008 USA Tel: 1 760 603 7200 Tel (Toll Free): 1 800 955 6288 Fax: 1 760 602 6500 E-mail: tech_service@invitrogen.com
MSDS Requests
To request an MSDS, visit our Web site at www.invitrogen.com. On the home page, go to `Technical Resources', select `MSDS', and follow instructions on the page. continued on next page
48
Technical Service, continued
Limited Warranty
Invitrogen is committed to providing our customers with high-quality goods and services. Our goal is to ensure that every customer is 100% satisfied with our products and our service. If you should have any questions or concerns about an Invitrogen product or service, contact our Technical Service Representatives. Invitrogen warrants that all of its products will perform according to specifications stated on the certificate of analysis. The company will replace, free of charge, any product that does not meet those specifications. This warranty limits Invitrogen Corporation's liability only to the cost of the product. No warranty is granted for products beyond their listed expiration date. No warranty is applicable unless all product components are stored in accordance with instructions. Invitrogen reserves the right to select the method(s) used to analyze a product unless Invitrogen agrees to a specified method in writing prior to acceptance of the order. Invitrogen makes every effort to ensure the accuracy of its publications, but realizes that the occasional typographical or other error is inevitable. Therefore Invitrogen makes no warranty of any kind regarding the contents of any publications or documentation. If you discover an error in any of our publications, please report it to our Technical Service Representatives. Invitrogen assumes no responsibility or liability for any special, incidental, indirect or consequential loss or damage whatsoever. The above limited warranty is sole and exclusive. No other warranty is made, whether expressed or implied, including any warranty of merchantability or fitness for a particular purpose.
49
Purchaser Notification
Introduction
Use of the MultiSite Gateway(R) Three-Fragment Vector Construction Kit is covered under the licenses detailed below.
Limited Use Label License No. 19: Gateway(R) Cloning Products
This product and its use is the subject of one or more of U.S. Patent Nos. 5,888,732, 6,143,557, 6,171,861, 6,270,969, and 6,277,608 and/or other pending U.S. and foreign patent applications owned by Invitrogen Corporation. The purchase of this product conveys to the buyer the non-transferable right to use the purchased amount of the product and components of the product in research conducted by the buyer (whether the buyer is an academic or for profit entity). The purchase of this product does not convey a license under any method claims in the foregoing patents or patent applications, or to use this product with any recombination sites other than those purchased from Invitrogen Corporation or its authorized distributor. The right to use methods claimed in the foregoing patents or patent applications with this product for research purposes only can only be acquired by the use of ClonaseTM purchased from Invitrogen Corporation or its authorized distributors. The buyer cannot modify the recombination sequence(s) contained in this product for any purpose. The buyer cannot sell or otherwise transfer (a) this product, (b) its components, or (c) materials made by the employment of this product or its components to a third party or otherwise use this product or its components or materials made by the employment of this product or its components for Commercial Purposes. The buyer may transfer information or materials made through the employment of this product to a scientific collaborator, provided that such transfer is not for any Commercial Purpose, and that such collaborator agrees in writing (a) not to transfer such materials to any third party, and (b) to use such transferred materials and/or information solely for research and not for Commercial Purposes. Notwithstanding the preceding, any buyer who is employed in an academic or government institution may transfer materials made with this product to a third party who has a license from Invitrogen under the patents identified above to distribute such materials. Transfer of such materials and/or information to collaborators does not convey rights to practice any methods claimed in the foregoing patents or patent applications. Commercial Purposes means any activity by a party for consideration and may include, but is not limited to: (1) use of the product or its components in manufacturing; (2) use of the product or its components to provide a service, information, or data; (3) use of the product or its components for therapeutic, diagnostic or prophylactic purposes; or (4) resale of the product or its components, whether or not such product or its components are resold for use in research. Invitrogen Corporation will not assert a claim against the buyer of infringement of the above patents based upon the manufacture, use or sale of a therapeutic, clinical diagnostic, vaccine or prophylactic product developed in research by the buyer in which this product or its components was employed, provided that none of (i) this product, (ii) any of its components, or (iii) a method claim of the foregoing patents, was used in the manufacture of such product. Invitrogen Corporation will not assert a claim against the buyer of infringement of the above patents based upon the use of this product to manufacture a protein for sale, provided that no method claim in the above patents was used in the manufacture of such protein. If the purchaser is not willing to accept the limitations of this limited use statement, Invitrogen is willing to accept return of the product with a full refund. For information on purchasing a license to use this product for purposes other than those permitted above, contact Licensing Department, Invitrogen Corporation, 1600 Faraday Avenue, Carlsbad, California 92008. Phone (760) 603-7200.
For additional information about Invitrogen's policy for the use and distribution of Gateway(R) Clone (R) (R) Distribution Policy Gateway clones, see the section entitled Gateway Clone Distribution Policy, page 52.
continued on next page
50
Purchaser Notification, continued
Limited Use Label License No. 23: GUS Control Vector Limited Use Label License No. 28: CMV Promoter
The GUS positive control vector in these products is claimed in patents and patent applications (See U.S. Patent No. 5,599,670 and Great Britain Patent No. 2,197,653) licensed to Invitrogen by Cambia Biosystems, L.L.C. ("CBL"). Use of the GUS gene is restricted to use as a positive control. Any other use may require a license from CBL. The use of the CMV promoter is covered under U.S. Patent Nos. 5,168,062 and 5,385,839 owned and licensed by the University of Iowa Research Foundation and is sold for research use only. Commercial users must obtain a license to these patents directly from the University of Iowa Research Foundation (UIRF), 214 Technology Innovation Center, Iowa City, Iowa 52242. For further information, please contact the Associate Director of UIRF, at 319-335-4546.
Limited Use Label License No. 48: araB Promoter
Products containing the araB promoter are the subject of U.S. Patent No. 5,028,530 and foreign equivalents and sold under patent license for research purposes only and are non-transferable. Inquiries for any commercial use, including production of material to be sold commercially or used in production or in product development efforts, which includes efforts toward regulatory approval, should be made directly to Xoma Corporation, 2910 Seventh Street, Berkeley, CA 94710, Tel: 1-510-644-1170 Fax: 1-510-6497571.
51
Gateway(R) Clone Distribution Policy
Introduction
The information supplied in this section is intended to provide clarity concerning Invitrogen's policy for the use and distribution of cloned nucleic acid fragments, including open reading frames, created using Invitrogen's commercially available Gateway(R) Technology. Invitrogen understands that Gateway(R) entry clones, containing attL1 and attL2 sites, may be generated by academic and government researchers for the purpose of scientific research. Invitrogen agrees that such clones may be distributed for scientific research by non-profit organizations and by for-profit organizations without royalty payment to Invitrogen. Invitrogen also understands that Gateway(R) expression clones, containing attB1 and attB2 sites, may be generated by academic and government researchers for the purpose of scientific research. Invitrogen agrees that such clones may be distributed for scientific research by academic and government organizations without royalty payment to Invitrogen. Organizations other than academia and government may also distribute such Gateway(R) expression clones for a nominal fee ($10 per clone) payable to Invitrogen. We would ask that such distributors of Gateway(R) entry and expression clones indicate that such clones may be used only for research purposes, that such clones incorporate the Gateway(R) Technology, and that the purchase of Gateway(R) ClonaseTM from Invitrogen is required for carrying out the Gateway(R) recombinational cloning reaction. This should allow researchers to readily identify Gateway(R) containing clones and facilitate their use of this powerful technology in their research. Use of Invitrogen's Gateway(R) Technology, including Gateway(R) clones, for purposes other than scientific research may require a license and questions concerning such commercial use should be directed to Invitrogen's licensing department at 760-603-7200.
Gateway(R) Entry Clones
Gateway(R) Expression Clones
Additional Terms and Conditions
52
Product Qualification
Introduction
This section describes the criteria used to qualify the components of the MultiSite Gateway(R) Three-Fragment Vector Construction Kit.
Vectors
The vectors are qualified as described below. pDONRTM vectors (pDONRTMP4-P1R, pDONRTMP2R-P3, pDONRTM221) * * * * * * * Structure of the vector is verified by restriction enzyme digestion. Functionality is verified in a 1 hour recombination assay with Gateway(R) BP ClonaseTM II enzyme mix. The ccdB gene is assayed by transformation using an appropriate E. coli strain. Structure of the vector is verified by restriction enzyme digestion. Functionality is verified in a 16 hour recombination assay with Gateway(R) LR ClonaseTM Plus enzyme mix. The ccdB gene is assayed by transformation using an appropriate E. coli strain. Structure of the vector is verified by restriction enzyme digestion.
pDESTTMR4-R3
pMS/GW
BP ClonaseTM II Enzyme Mix LR ClonaseTM Plus Enzyme Mix
Gateway(R) BP ClonaseTM II enzyme mix is functionally tested in a 1 hour recombination reaction followed by a transformation assay. Gateway(R) LR ClonaseTM Plus enzyme mix is functionally tested in a 16 hour MultiSite Gateway(R) LR recombination reaction followed by a transformation assay.
One Shot(R) TOP10 Chemically Competent E. coli
1.
One Shot(R) TOP10 chemically competent cells are tested for transformation efficiency using the control plasmid included in the kit. Transformed cultures are plated on LB plates containing 100 g/ml ampicillin and the transformation efficiency is calculated. Test transformations are performed in duplicate. Transformation efficiency should be greater than 1 x 109 cfu/g plasmid DNA. To verify the absence of phage contamination, 0.5-1 ml of competent cells are added to LB top agar and poured onto LB plates. After overnight incubation, no plaques should be detected. Untransformed cells are plated on LB plates containing 100 g/ml ampicillin, 25 g/ml streptomycin, 50 g/ml kanamycin, or 15 g/ml chloramphenicol to verify the absence of antibiotic-resistant contamination.
2.
3.
53
Glossary of Terms
attL, attR, attB, and attP
The recombination sites from bacteriophage lambda that are utilized in the Gateway(R) Technology. * attL always recombines with attR in a reaction mediated by the LR ClonaseTM II enzyme mix (for standard Gateway(R) reactions) or LR ClonaseTM Plus enzyme mix (for MultiSite Gateway(R) reactions). The LR reaction is the basis for the entry clone(s) x destination vector reaction. Recombination between attL and attR sites yields attB and attP sites on the resulting plasmids. attB sites always recombine with attP sites in a reaction mediated by the BP ClonaseTM II enzyme mix. The BP reaction is the basis for the reaction between the donor vector (pDONRTM) and PCR products or other clones containing attB sites. Recombination between attB and attP sites yields attL and attR sites on the resulting plasmids.
*
BP ClonaseTM II Enzyme Mix ccdB Gene
A proprietary mix of lambda recombination proteins that mediates the attB x attP recombination reaction. A gene that encodes a protein that interferes with E. coli DNA gyrase, thereby inhibiting the growth of standard E. coli hosts. This gene is present on Gateway(R) destination, donor, and supercoiled entry vectors. When recombination occurs between a destination vector and an entry clone, the gene of interest replaces the ccdB gene. Cells that take up unreacted vectors carrying the ccdB gene, or byproduct molecules that retain the ccdB gene, will fail to grow. This allows highefficiency recovery of only the desired clones.
Destination Vector Gateway(R)-adapted expression vectors which contain attR sites and allow
recombination with entry clones.
Donor Vector (pDONRTM)
A Gateway(R) vector containing attP sites. This vector is used for cloning PCR products and DNA sequences of interest flanked by attB sites (expression clones) to generate entry clones. When PCR fragments modified with attB sites are recombined with the pDONRTM vector in a BP reaction, they yield an entry clone. PCR fragment (attB sites) + pDONRTM vector (attP sites) entry clone
Entry Clone
The result of cloning a DNA segment into an entry vector or donor vector. For MultiSite Gateway(R) applications, the entry clone contains the DNA sequence of interest flanked by attL sites or a combination of attL and attR sites. The entry clone can be used for subsequent transfers into destination vectors.
Entry Vector (pENTRTM)
A Gateway(R) vector containing attL1 and attL2 sites used for cloning DNA fragments using either TOPO(R) Cloning or conventional restriction enzymes and ligase. continued on next page
54
Glossary of Terms, continued
Expression Clone
The result of subcloning the DNA of interest from an entry clone into a destination vector of choice by LR recombination. For MultiSite Gateway(R) applications, the expression clone contains DNA fragments transferred from multiple entry clones into a single destination vector. Each DNA fragment of interest in the expression clone is flanked by attB sites. Entry clone(s) + destination vector expression clone
Gateway(R) Technology
A universal cloning method based on the site-specific recombination properties of bacteriophage lambda (Landy, 1989) that allows highly efficient transfer of a DNA sequence of interest into multiple vector systems.
LR ClonaseTM Plus Enzyme Mix
A proprietary mix of lambda and E. coli recombination proteins that mediates the attL x attR recombination reaction. This enzyme has been optimized for demanding applications including MultiSite Gateway(R), but is also suitable for use in standard Gateway(R) applications.
MultiSite Gateway(R) Technology
An extension of the Gateway(R) Technology that facilitates simultaneous cloning of multiple DNA fragments in a defined order and orientation.
55
References
Bernard, P., and Couturier, M. (1992). Cell Killing by the F Plasmid CcdB Protein Involves Poisoning of DNA-Topoisomerase II Complexes. J. Mol. Biol. 226, 735-745. Hartley, J. L., Temple, G. F., and Brasch, M. A. (2000). DNA Cloning Using in vitro Site-Specific Recombination. Genome Research 10, 1788-1795. Kozak, M. (1987). An Analysis of 5-Noncoding Sequences from 699 Vertebrate Messenger RNAs. Nucleic Acids Res. 15, 8125-8148. Kozak, M. (1991). An Analysis of Vertebrate mRNA Sequences: Intimations of Translational Control. J. Cell Biology 115, 887-903. Kozak, M. (1990). Downstream Secondary Structure Facilitates Recognition of Initiator Codons by Eukaryotic Ribosomes. Proc. Natl. Acad. Sci. USA 87, 8301-8305. Landy, A. (1989). Dynamic, Structural, and Regulatory Aspects of Lambda Site-specific Recombination. Ann. Rev. Biochem. 58, 913-949. Orosz, A., Boros, I., and Venetianer, P. (1991). Analysis of the Complex Transcription Termination Region of the Escherichia coli rrnB Gene. Eur. J. Biochem. 201, 653-659. Ptashne, M. (1992). A Genetic Switch: Phage (Lambda) and Higher Organisms (Cambridge, MA: Cell Press). Shine, J., and Dalgarno, L. (1975). Terminal-Sequence Analysis of Bacterial Ribosomal RNA. Correlation Between the 3'-Terminal-Polypyrimidine Sequence of 16-S RNA and Translational Specificity of the Ribosome. Eur. J. Biochem. 57, 221-230. Trinh, T., Jessee, J., and Bloom, F. R. (1994). STBL2: An Escherichia Coli Strain for the Stable Propagation of Retroviral Clones and Direct Repeat Sequences. FOCUS 16, 78-80.
(c)2002-2004 Invitrogen Corporation. All rights reserved.
For research use only. Not intended for any animal or human therapeutic or diagnostic use.
56
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