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PCR failure from yeast genomic DNA

pcr yeast genomic dna

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#1 supermr@tamu



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Posted 03 February 2012 - 11:13 AM

Howdy, all,

I got a problem here... Plz help...Posted Image
I tried to delete a gene in baker's yeast by homologous recombination and need to confirm the gene replacement by PCR. I transformed yeast with the deletion cassette and a couple of candidates showed up and I picked up 6 to test for deletion.
Our group always isolate genomic DNA and do PCR to test. Normally I grow cell in rich medium YPD to isolate the genomic DNA and it worked for me almost every time.
But this time there is a plasmid I need to maintain so I grew candidate cells in selective minimal medium. I used 5ml overnight minimal medium culture for genomic DNA isolation (phenol/chloroform and glass beads vortex method), everything looked fine - I got some white DNA pellets and washed once with 70% ethanol and then dissolved in dd water.
Then I did PCR on these obtained 6 genomic DNAs along with a positive control (WT genomic DNA isolated from YPD culture). Weird thing is, the positive control showed a nice band at right size but no any band showed up for all my 6 candidate genomic DNA!!

This is the second time I observe this problem. Last time I had the same problem but after growing cells in YPD I got successful results. But this time, as I mentioned, I need selective minimal medium to maintain a plasmid (with essential gene on it).

I was careful to make sure no phenol phase was transferred, so it should not be phenol problem. I should have isolated enough genomic DNA as I could see a good amount of DNA white pellet after ethanol precipitation.

Anybody had similar experience? Probably some Taq inhibitors were carried over (somehow not a problem for YPD culture)? If so, what is the best way to remove them?

Thanks in advance.

#2 Trof


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Posted 03 February 2012 - 03:07 PM

Put some artificial DNA and primers for it to your samples test if it's inhibition. They could also be simply negative.

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