[Drosophila] technique suddenly not working

[Marc S. Halfon]

Hi Kathleen--
We ran into some similar problems recently and it turned out to be the 
PCR mix--we had switched to Strategene's pfu turbo and suddenly started 
having trouble off the single-fly preps. The same prep worked fine with 
Qiagen HotStar taq. Our guess is that the pfu is pickier about salt 
conditions etc., as it works great off of more purified genomic DNA. So 
you might want to try either (1) switching Taqs or (2) cleaning up the 
DNA first.
Marc


On Nov 11, 2005, at 5:01 PM, Kathleen Gajewski wrote:

> I'm suddenly having trouble with the Gloor and Engels technique for 
> PCR from single fly genomic preps (I'm trying to work out a PCR test 
> for verifying transgenic lines).  I've remade my solutions- no luck.  
> I've included fly strains/primer combinations/PCR conditions that have 
> worked in the past-still no luck.  I've included positive controls of 
> the purified construct DNA, and those work just fine, so I know the 
> PCR itself is not at fault.  Does anyone have some trouble shooting 
> advice??  Usually the technique works just fine,  but every now and 
> then it just fails out of the blue and I'm at my wits end with this!!
>
> Thanks
>
> Kathleen
>
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-----------------------------------
Marc S. Halfon, Ph.D.
Assistant Professor
Dept. of Biochemistry and Center of Excellence in Bioinformatics
SUNY at Buffalo