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failure of PCR from yeast genomic DNA

PCR yeast genomic dna

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#1 supermr@tamu

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Posted 03 February 2012 - 11:09 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 akhshik

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Posted 03 February 2012 - 10:22 PM

welcome to the world of molecular biology, do not think that you have inhibition for Taq or any thing bad happens to your DNA it is normal to see this kind of result, and I promise your Yeast does not have your expected sequence, either it is not transformed or lost the sequence in minimal medium,
just repeat the experience like the first time that you had the positive PCR: YPD culture not minimal.

Cheers,
Akhshik.





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