Non-lethal genotyping

A forum for sharing and discussing experimental protocols.

Non-lethal genotyping

Postby speese » Fri Sep 02, 2011 3:01 pm

I was wondering if anyone has consistent results with the wing-clip non-lethal genotyping protocol that was published in '09. I have been trying to get this established in the lab and I have been getting some variable results. In particular, if you have good primer sets for detecting common transgenes (Gal4, UAS, Gal80, GFP etc.) I would be interested in those sequences. I have been using Primer3 to design my oligos, but I still have been having a lot of trouble getting good sets of oligos that amplify efficiently. The primer sets work great against isolated plasmid containing the appropriate transgenes, but once I go to use them on genomic they fail or amplify poorly. I have ruled out PCR inhibition as a cause, as I am able to spike the plasmid into the genomic and get good amplification. Any thoughts from groups who consistently use this technique would be greatly appreciated.
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