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Ethanol precipitation problem !


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#1 greengumbo

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Posted 29 November 2009 - 04:42 PM

Hi,

In the past I have carried out heaps of phenol/chloroform clean ups of DNA but this morning....probably due to an excess of coffee....I made a bit of a blunder and I'm hoping all you clever people can help me out.

Basically I have taken a 100ul restriction enzyme-treated plasmid DNA sample through a Phenol then phenol/chloroform then chloroform clean up. Unfortunately, instead of removing the aqueous layer after the final spin (chloroform and my DNA) I added the 100% Ethanol straight to the whole lot....doh ! I must now have in my sample 100ul DNA / 100ul Chloroform and 200ul 100% EtOH. My initial thought is to add more ethanol and place it in the -80'C freezer, leave for a while and then spin it down and hope to precipitate out the DNA......will this work ? Would adding Na Acetate help ?

Cheers in advance for the help !

GG

#2 phage434

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Posted 29 November 2009 - 05:48 PM

I'd be concerned that the ethanol would mix in both the water and chloroform layers. If this was a throw away sample, I'd just do it again. If a precious sample, I would add substantially more chloroform (perhaps 1-2 ml) and some more water, diluting the ethanol, spin, and recover the aqueous layer. You could extract with water a second time to get residual DNA. Then I'd precipitate as normal (perhaps with some pellet paint or glycogen to help things along due to the low DNA concentrations).




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