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BD GenomeWalker™ Universal Kit User Manual
V. BD GenomeWalker DNA Walking continued
18. Commence cycling in a DNA Thermal Cycler 480 (PE Biosystems),
using the following two-step cycle parameters:
• 5 cycles:
94°C
72°C
25 sec
3 min
• 20 cycles:
94°C
67°C
25 sec
3 min
• 67°C for an additional 7 min after the final cycle.
Note: Do not use a three-step cycling program (e.g., 95°C melting, 60°C annealing,
68°C extension). See Appendix C for cycling parameters for the GeneAmp PCR
Systems 2400 and 9600.
19. Analyze 5 µl of the secondary PCR products on a 1.5% agarose/EtBr
gel, along with DNA size markers such as a 1 kb ladder or λ/Hind III
digest. If you do not see any product, perform four additional cycles.
Store the unused portion of each secondary PCR at 4°C until you have
confirmed that the procedure has been successful. At that point,
proceed with analyzing and cloning the fragments of interest (e.g.,
putative promoter fragments), as described in Section VII.
BD Biosciences Clontech
18
www.bdbiosciences.com
Protocol No. PT3042-1
Version No. PR47605