Phenol purification of genomic DNA degraded by RNA? - (Feb/06/2006 )

I did a genomic preparation of my strains of interest and when I ran them on gel, they were obvoiusly degraded andcontaminated by RNA. Should I re-do the genomic preparation or can I just do a phenol purification of the genomic DNA I already have?

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Are you saying the DNA is degraded? If its degraded then you can't salvage the DNA by a phenol/chloroform extraction. I would re-do the extraction. You can add Rnase A after the lysis step and incubate for 20 mins or so at room temperature to get rid of RNA.

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Thanks! My mentor also told me to take 1ul of my genomic prep and dissolve in 10ul TE buffer and leave overnight at 37 degrees celsius and run on a gel the next day and that I should see no degradation (this was the case for other 2 genomics i made before these ones), and I was wondering, why 37 degrees?

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that doesn't make any sense to me...did he give you a reason with some chemistry behind it?

sometimes genomic DNA is pretty viscous and it can be difficult to fully dissolve, but if your DNA is degraded I don't see how it can be recovered by that means?

I am just a grasshopper here and there may certainly be something that I am missing, but I would think it would be wise to repeat the prep like ML1975 says, and take extra precaution not to shear your DNA during the steps

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Sounds to me like what you are seeing in the gel is only the degraded RNA?? By keeping the sample at 37C o/n you allow the RNA to be completely degraded. Another way to approach this would be to add RNAse directly as suggested here... I think you would still incubate at 37C b/c the enzyme likes this temperature, but you wouldn't have to wait so long...depending on amount 5-15 minutes should be enough...

If your DNA is already degraded this will not help you, you can't put it back together and you would have to start over...

HTH

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