The gateway cloning system
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How to generate an entry clone. The Gateway® Cloning System. Contents. Options for entering the Gateway ® system Defining the BP Clonase ™ reaction Defining the LR Clonase ™ reaction Description of Ultimate ™ ORF collection

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The Gateway® Cloning System

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The gateway cloning system

How to generate an entry clone

The Gateway® Cloning System

Contents

Options for entering the Gateway® system

Defining the BP Clonase™ reaction

Defining the LR Clonase™ reaction

Description of Ultimate™ ORF collection

Description of Vector NTI Advance™ software for in silico cloning


The gateway reactions

The Gateway® Reactions

Invitrogen Proprietary & Confidential


Different ways to generate the entry clone

L1

L2

L1

L2

+

B1

Gene

+

Gene

digested Entry Vector

TOPO-Activated

Entry Vector

digested DNA Fragment

PCR Product

P1

P2

ccdB

+

B1

Gene

B2

Donor Vector

PCR Product

attB

Different ways to generate the entry clone

BP Cloning

TOPO® Cloning

BP Clonase™

TOPO®

Gene

L1

L2

Entry Clone

Ligase

4. Pre-made entry clone

5. Custom-made entry clone

Restriction/Ligase Cloning

L1

L2

ORF

ORF Collection

Invitrogen Proprietary & Confidential


Bp cloning the reaction

90-99% correct clones

on Kan plates

BP Cloning – The Reaction

+

+

BP Clonase™

Invitrogen Proprietary & Confidential


Bp cloning primer design for pcr

Gene Specific

Primer Sequence

BP Cloning - Primer Design for PCR

  • GGGG and the attB1 sequence must be added to the 5’-primer (sense)

  • GGGG and the attB2 sequence must be added to the 3’-primer (antisense)

    attB1

    5’ – GGGGACAAGTTTGTACAAAAAAGCAGGCTNNN…

    attB2

    5’ – GGGGACCACTTTGTACAAGAAAGCTGGGTNNN…

Invitrogen Proprietary & Confidential


Bp cloning some examples

BP Cloning – Some Examples

Correct

PCR DNA

PCR DNA

Colonies/l

Size (kb)

Clones/Total

(fmol)

(ng)

Transformation

Clones Examined

15

3

1223

0.26

10/10

38

7.5

2815

15

10

507

1.0

49/50

38

25

1447

15

14

271

1.4

48/50

38

35

683

15

34

478

3.4

9/10

38

85

976

15

46

190

4.6

10/10

38

115

195

15

69

30 (235)*

6.9

47/50

38

173

54 (463)*

7.5

50.5

16 (112)*

10.1

15/16

37.5

252.5

42 (201)*

*After overnight incubation

Invitrogen Proprietary & Confidential


Bp cloning rt pcr using att b containing primers

BP Cloning – RT-PCR Using attB-Containing Primers

Tyrosine

Kinase

Transferrin

Receptor

Target Template:

EIF4e

b-Adaptin

MAP4

+ + - -

+ + - -

+ + - -

+ + - -

+ + - -

attB-containing primers:

Platinum® Taq DNA Polymerase

Platinum® Taq DNA Polymerase High Fidelity

Platinum® Pfx DNA Polymerase

Total RNA isolated from HeLa cells, first strand cDNA synthesized using THERMOSCRIPT™ RT.


Topo cloning topo ta

TOPO® Cloning -TOPO®TA

Invitrogen Proprietary & Confidential


Topo cloning directional topo

TOPO® Cloning – Directional TOPO®

Invitrogen Proprietary & Confidential


Restriction ligase cloning

Restriction/Ligase cloning

Use when there are convenient sites to cut insert out of another plasmid

Must cut out ccdB gene by using one of four RE sites flanking the ccdB

Reading frame of insert must be considered, as well as downstream expression elements

Various reading frames of pENTR vectors are available

Invitrogen Proprietary & Confidential


Pre existing orf collection

Pre-existing ORF collection

Invitrogen’s Ultimate™ ORF collection

16,272 human ORFs (Oct 2006 release)

Amber stop codons

Sequence verified

Ready to use in LR reactions

http://orf.invitrogen.com/cgi-bin/ORF_Browser

Invitrogen Proprietary & Confidential


5 custom gene synthesis

5. Custom Gene Synthesis

  • Quick and cost-effective

  • No PCR amplification necessary

  • 100% accuracy (sequence verified)

  • Optional codon optimization for expression

Invitrogen Proprietary & Confidential


In silico cloning using vector nti advance tm 10 3

In silico cloning using Vector NTI AdvanceTM 10.3

DNA of interest

Primers for PCR reaction

Cloning Strategy

Invitrogen Proprietary & Confidential


Gateway summary

Gateway® Summary

Invitrogen Proprietary & Confidential


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