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BD GenomeWalker™ Kits User Manual
I. Introduction continued
BD GenomeWalker™ "Libraries"
Amplify gene of interest
from all four libraries
Genomic DNA
fragment
No binding site for AP1
Binding site is created
on template of interest
by extension of GSP1
GSP2
GSP1
5' –
N
– 5'
N
AP1
AP2
BD GenomeWalker
Adaptor
AP1
GSP1
Primary PCR with BD Advantage™ 2
Polymerase Mix (contains BD TaqStart™ Antibody)
AP2
GSP2
Secondary or "nested" PCR with
BD Advantage™ 2 Polymerase Mix
Examine products on
an agarose/EtBr gel
M 1
2
3
4
M
3.0
2.0
1.6
1.0
0.5
• Clone & characterize major PCR products
• Test for promoter activity by cloning into reporter vector
Figure 1. Flow chart of the BD GenomeWalker™ protocol. The gel shows typical results
generated by nested PCR with the BD GenomeWalker human libraries and gene-specific primers.
Primary and secondary (nested) PCR was performed using BD Advantage 2 Polymerase Mix and
the cycling parameters described in the protocol. Lane 1: EcoR V Library. Lane 2: Dra I Library.
Lane 3: Pvu II Library. Lane 4: Ssp I Library. Lane M: DNA size markers. The absence of a major
product in one of the libraries is not unusual. In our experience, there is no major band in one or more
lanes in approximately half of the BD GenomeWalker experiments. As explained in the Expected
Results and Troubleshooting Guide (Section V), this is usually because the distance between the
primer and the upstream restriction site is greater than the capability of the system. N: Amine group
that blocks extension of the 3' end of the adaptor-ligated genomic fragments. AP: Adaptor primers.
GSP: Gene-specific primers.
BD Biosciences Clontech
4
www.bdbiosciences.com
Protocol No. PT1116-1
Version No. PR47603