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Lambda Red verification PCR trouble

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#1 Sindit



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Posted 14 March 2011 - 08:53 AM

Hello everyone! Hope someone know how to solve this drama:

I followed a protocol on lambda red knockout( http://openwetware.o...ene_replacement ), to delete a gene and insert chloramphenicol resistance into e. coli.

After eletroporation, the cells growth in LB+antibiotic (25ug/ml) as expected, but into PCR verification I got the expected band (500pb) for the original e.coli sample, and no band into the mutant sample, where I wanted to see a 1100/1200pb... I'm using primers as described into the procedure above: the same 25pb flankers used into the deletion cassete.

Negative control is ok(clear), and sometimes appears into mutant samples 2 very soft bands into aprox. 480pb and 750pb, but I can't consider this an amplification, since it's not strong enough compared to the non-electroporated e. coli, it's just a "ghost" into the gel. Primer design is not good also (because of the lambda red protocol), but in my opnion doesn't make sense to change it because worked pretty fine into the non-mutant. (I already tried a different primer pair, same result...)

Feel free to express some idea on the pcr, any other way to verify the lambda red then the PCR are welcome.

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