yeast genomic DNA extraction - (Mar/13/2007 )

Hi

I'm having some trouble extracting gDNA from S. cerevisiae and a collegue of mine is having the same problem with C. albicans. We both use a kit and follow all the instructions but then, when we run the samples on agarose the DNA band doesn't correspond with our expectation according to the spectofotometer quantifications.

Our yields are great and 260/280 ratios are between 1.8-2.0. We already checked the spectofotometer with salmon sperm with stander concentration but it's everything ok with the equipment. There must be something in the extraction...

and if we go on with PCR, enzyme digestions or even random priming labeling, these procedures don't not work/have very low yields.

Suggestions?

Thks!

is your DNA free from RNA?

-Agarose the DNA band doesn't correspond with our expectation according to the spectofotometer quantifications.

a common cause is RNA contamination.. which can cause the spectophotometer reading to vary somewhat. The agarose band is the real amount of DNA.

As for DNA not cutting with Restriction enzymes... the cause is that the DNA is not clean enough. Conduct 2 - 3 phenol chloroform extractions. (when appropriate resuspend DNA in a volume of 500ul, as you can lose alot of volume during the extraction) Once cleaned try cutting with EcoRI. If it fails to cut with EcoRI, conduct more phenol/chlorofom extrations. EcoRI, can be considered one of the best enzyme around. If it can't cut your DNA, nothing can.

As the gDNA is no where as clean as plasmid DNA, give it more time to digest. Also pay attention to DNA degredation by nucleases. (ie the digestion smear starts centering on very small fragments around 500bp.) Since you are experiencing the above problems, losing your DNA by nuclease reactivation in digestion buffer is a real danger.

As for PCR, again the DNA is not clean enough. And in my hands, even when 'cleaned', this is still not good enough. I dilute my a sample of genomic DNA at least 1:5 to 1:50 with sterile distilled water to serve as a template. Dilution help dilute away any remaining contaminant. And PCR amplifies the weak signal. Do note that genomic DNA kept in sd water does live long... about 5-6 months in my hands. For long term storage always keep the DNA in TE.

But with all said, it takes a little practice to get nice clean DNA. Be brave and poke around with your protocol see if it can be improved. If it can't, see what extra things you can do to the DNA once you have it.

About the RNA contamination... It can be although I always check the Ph:Ch pH before starting the DNA extraction so that I'm having DNA instead of RNA.

The enzyme I'm using is cutting DNA, I can see the expected smear in the gel but again with lower intensity as I expected. I already tried digesting it overnight but the results are the same.

Thanks for the suggestions, I'll go on trying...