Gateway cloning recombination-based cloning method comprised of ...

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Benefits of Gateway - an example. - testing epitope tags. ORF ... attL2. ccdB/cam. attR1. attR2. ORF. attL1. attL2 myc- cherry- egfp-. ORF. B1. B2. ORF. B1. B2.
Gateway cloning recombination-based cloning method comprised of two separate reactions BP reaction attB1

ORF

LR reaction attB2

attL1

BP clonase

ORF

attL2

LR clonase

attP1

ccdB/cam

attP2

attR1

ccdB/cam

attR2

attL1

ORF

attL2

attB1

ORF

attB2

from the Lawson Lab website

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Benefits of Gateway - an example - testing epitope tags

attB1

PCR amplify with attB containing primers

ORF attB2

ORF

attB1

attP1

attB2

ccdB/cam

attP2

ORF

attL2

generate entry clone with BP reaction

pDONR221

attL1

from the Lawson Lab website

sequence verify!!

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Rapid simultaneous generation of epitope tagged injection constructs

attL1

ORF

attL2

attR1 ccdB/cam attR2

B1

ORF

B2

attL1

ORF

myc- attR1 ccdB/cam myc- B1

ORF

attL2

attR2

B2

attL1

ORF

egfp- attR1 ccdB/cam egfp-B1

ORF

attL2

attL1

ORF

attR2 cherry- attR1 ccdB/cam attR2

B2

cherry-B1

ORF

requires availability of appropriate Destination vectors!!

from the Lawson Lab website

attL2

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B2

pCS-based Destination constructs - N terminal epitope tags

pCSDest CMV

SP6

attR1 ccdB/cam attR2

polyA

T7

pCSMTDest CMV

SP6

6xmyc attR1 ccdB/cam

attR2

polyA

T7

pCSegfpDest CMV

SP6

egfp

attR1 ccdB/cam attR2

T7

polyA

cherry

attR1 ccdB/cam attR2

T7

polyA

pCScherryDest CMV

SP6

from the Lawson Lab website

4

Validation of pCS-based Destination vectors

LR reactions

pENTRN3icd x pCSMTDest

pENTRN3icd x pCSmCherryDest

pENTRN3icd x pCSegfpDest

pENTRegfp2 x pCSmCherryDest

from the Lawson Lab website

5

pCS-based Dest plasmid for generating C-terminal fusion

attB1

ORF (no stop codon) attB2

attB1

ORF (no stop codon)

attB2

attP1

ccdB/cam

attP2

pDONR221

attL1

ORF (no stop codon)

PCR amplify with attB containing primers

Perform BP reactions with PCR product and appropriate DONR vector

attB2

C-term tag attB3

attB2

C-term tag

attB3

attP2r

ccdB/cam

attP3

pDONRP2r-P3

attL2

attR2

from the Lawson Lab website

C-term tag

attL3

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Perform two-way multisite Gateway reaction to generate C-term epitope tagged injection construct attL1

ORF (no stop codon)

attL2

attR2

CMV SP6 pCSDest-2

CMV

SP6

B1

attR1

ccdB/cam

ORF (no stop codon)

B2

C-term tag

attR3

C-term tag

from the Lawson Lab website

T7

B3

attL3

polyA

T7

polyA

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Validation of pCSDest2

from the Lawson Lab website

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Gateway compatible Tol2 vectors pTolDest EcoRV

Tol2

attR1 ccdB/cam attR2

polyA

T7

Tol2

pTolMTDest Tol2

EcoRV

6xmyc attR1 ccdB/cam

attR2

T7

Tol2

polyA

pTolegfpDest EcoRV

Tol2

egfp

attR1 ccdB/cam attR2

T7

polyA

Tol2

polyA

Tol2

pTolcherryDest EcoRV

Tol2

cherry

attR1 ccdB/cam attR2

from the Lawson Lab website

T7

9

Validation of Tol-based Destination vectors pTol-lmo2-Dest x pENTRegfp2

pTol-lmo2-egfpDest x pENTRdsRedEx

pTol-lmo2-mCherryDest x pENTRegfp2

from the Lawson Lab website

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Three-way multisite to generate C-term fusions driven by a tissue specific promoter attL1

ORF (no stop codon)

attL2

C-term tag

attR2

promoter

attL4

Tol2

attR4

attL3

attR1

ccdB/cam

attR3

T7

polyA

Tol2

pDest Tol pA

Tol2

B4

promoter

B1

ORF (no stop codon)

B2

C-term tag

from the Lawson Lab website

B3

T7

polyA 11

Tol2

Validation of 3-way Gateway construct

from the Lawson Lab website

12