The Gateway

1
The Gateway Cloning System
How 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

2
The Gateway Reactions
3
Different ways to generate the entry clone

1. TOPO Cloning

1. BP Cloning

TOPO
BP Clonase
L2
Gene
L1
Entry Clone
Ligase
4. Pre-made entry clone 5. Custom-made entry clone

1. Restriction/Ligase Cloning

L2
ORF
L1
ORF Collection
4
BP Cloning The Reaction


BP Clonase
5
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

6
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
7
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.
8
TOPO Cloning -TOPOTA
9
TOPO Cloning Directional TOPO
10
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

11
Pre-existing ORF collection
16,272 human ORFs (Oct 2006 release) Amber stop
codons Sequence verified Ready to use in LR
reactions
Invitrogens Ultimate ORF collection
http//orf.invitrogen.com/cgi-bin/ORF_Browser
12
5. Custom Gene Synthesis

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

13
In silico cloning using Vector NTI AdvanceTM 10.3
DNA of interest
Primers for PCR reaction
Cloning Strategy
14
Gateway Summary