[Drosophila] FRT-directed deletions PCR genotyping?

[Zhang Yi]

2008-08-07

Do anybody have experience on making FRT-directed deletions using Exelixis stocks described in Park et al 2004 Nat.Genetics paper? I did one WH minus - RB plus deletion, but failed to find any correct deletion in 96 lines via PCR. My PCR was using similar primers described in Park et al paper. I did 20ul rxn with Takara LA Taq. Annealing temperature touchdown from 68c, 65c, 62c, 59c (each 2 cycle) then 56c 25 cycles. I used 1 min annealing and 72c, 12min extension. The product should be ~7.3kb according to the paper, but I failed to get any band in between primer and the well. However there were thick bands in the well. 

Could anybody help me to figure out what is the problem? Thanks a lot!

Best,
Yi
 				
-- 
No fate but what we make.

Zhang Yi
Lab of Neuroscience, B109
National Institute of Biological Sciences,
7 Kexueyuan Rd. ZGC Life Sciences Park,
Beijing 102206
China, People's Republic of
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