The Gateway® Cloning System How to generate an entry clone Contents Options for entering the Gateway ® system Defining the BP Clonase reaction Defining.

The Gateway® Cloning SystemHow to generate an entry clone

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

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The Gateway® Reactions

LR clonase

++BP clonase

attL1 attL2

EntryClone

KanR

ExpressionClone

attB1 attB2

AmpR

attR1 attR2

ccdB

DestinationVector

AmpR

attP1 attP2

ccdB

DonorVector

KanR

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Different ways to generate the entry clone

2. TOPO® Cloning

TOPO®BP Clonase™

1. BP Cloning

PCR Product

+TOPO-ActivatedEntry Vector

L1 L2

Gene+attB PCR Product

B2GeneB1Donor Vector

P2ccdBP1

Entry Clone

L2GeneL1

+digested DNA Fragment

GeneB1digested Entry Vector

L2L1

4. Pre-made entry clone5. Custom-made entry clone

Ligase

3. Restriction/Ligase Cloning

ORF Collection

L2ORFL1

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1. BP Cloning – The Reaction

90-99% correct cloneson Kan plates

geneattB1 attB2

+attP1 attP2

ccdB

D o norVector

K anR

+

gene

attL1 attL2

EntryC lone

K anRccdBattR1 attR2

BP Clonase™

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1. 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…

Gene SpecificPrimer Sequence

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*After overnight incubation

1. BP Cloning – Some Examples

Size (kb) PCR DNA(fmol)

PCR DNA(ng)

Colonies/lTransformation

CorrectClones/Total

Clones Examined

0.261538

37.5

12232815

10/10

1.01538

1025

5071447

49/50

1.41538

1435

271683

48/50

3.41538

3485

478976

9/10

4.61538

46115

190195

10/10

6.91538

69173

30 (235)*54 (463)*

47/50

10.17.5

37.550.5252.5

16 (112)*42 (201)*

15/16

TyrosineKinase

Transferrin ReceptorTarget Template: EIF4e b-Adaptin MAP4

attB-containing primers: + + - - + + - - + + - - + + - - + + - -

Platinum® Pfx DNA Polymerase

Platinum® Taq DNA Polymerase

High Fidelity

Platinum® Taq DNA

Polymerase

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

1. BP Cloning – RT-PCR Using attB-Containing Primers

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2. TOPO® Cloning -TOPO®TA

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2. TOPO® Cloning – Directional TOPO®

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3. 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

pENTR™ 1A Vector in reading frame 0

pENTR™ 2B Vector in reading frame +1

pENTR™ 3C Vector in reading frame +2

pENTR™ 4 Vector in reading frame 0; modified polylinker from 1A

pENTR™ 11 Contains both an E. coli (SD) and eukoryotic (Kozak) ribosome binding site

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4. Pre-existing 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’s Ultimate™ ORF collection

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5. Custom Gene Synthesis

• Quick and cost-effective• No PCR amplification necessary• 100% accuracy (sequence verified)• Optional codon optimization for

expression

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In silico cloning using Vector NTI AdvanceTM 10.3

Primers for PCR reaction

Cloning Strategy

DNA of interest

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Gateway® Summary

Gene

Gene

Entry C lone

O RFcollection

PC R Library

Genesynthesis

YourSource