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Identifying various unknown P-elements in Drosophilia


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#1 David_K

David_K

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Posted 28 September 2010 - 12:27 PM

So,

Long story short, our collection of mutants got their labels mixed up, and now I need to identify the site of mutations. This collection of flys was a mishmash of various P-elements - aka pBac, P{XP} Mi{ET} etc.

I've tried internal PCR using pBac and p{XP} primers, but have come up fairly empty handed.

Part of the problem is that we have a lot of random DNA floating around in the lab, and with 40 x 2 PCR amplifications of the nested PCR along with the sequencing reaction, I'm not sure that I'm not amplifying random tidbits that are floating around.

I would like to restart with the genomic prep of the flies, and then try to identify the type of P-element first before proceeding to PCR/sequencing.

Are there any good ways of going about doing this?




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