Trouble with inconsistant DNA extraction - (Dec/01/2008 )

Hello all

I'm trying to extract genomic DNA from rat bone marrow after digesting it for various times with an endonuclease. But I'm having really inconsistant DNA recovery. The pictures below show a good one and bad one. It seemed to work okay about a month ago, but now I'm getting really poor results.

I'm doing a phenol:chloroform:isoamyl alcohol extraction, followed by two water saturated ether ether extraction, and then the ethanol precipitation overnight in a freezer.

Can anyone give me some ideas of what I could be doing wrong??


Thanks.

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Heres the bad picture

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Did you stop the RE reaction? Just asking

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I assume you mean the nuclease digestion? Then yes. The digestion looks fine to me. I've screwed up before and put in too much nuclease and ended up with just a massive single band of small fragments, which doesn't occur on the ones that I'm having trouble with.


So I'm pretty positive it's the DNA extraction.

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Anyone have any suggestions of common mistakes I could be doing in DNA extraction?

I could really use some help....

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