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removal of ethanol in PCR product purification


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#1 harry348

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Posted 24 May 2013 - 07:56 AM

I am using a qiagen pcr purification kit to purify my pcr produtcs. My problem here is at the last of purification i forgot to centrifuge the column for the second time after adding ethanol to the column so that i can remove all the ethanol that is present. So missing this step i added MQ water to the coulmn and eluted in 30ul.

Now how can i remove this ethanol from the elute ??? After this step i placed elute at 4C.

Please give me some ideas to remove ethanol

thnax in advance

harry

#2 Curlis

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Posted 24 May 2013 - 08:46 AM

Heat it at 37 degree for 1 hr. I think that will be more than sufficient.

#3 harry348

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Posted 24 May 2013 - 08:48 AM

Thanx ashu, but what about he DNA in it?

#4 Spudgun

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Posted 26 May 2013 - 01:10 AM

Thanx ashu, but what about he DNA in it?


What temp. is your body at?

#5 Trof

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Posted 26 May 2013 - 05:50 AM

I'm not really sure that this small amount of ethanol (i.e. highly diluted) would preferentially evaporate.
To be completely sure (albeit with the risk of losing some amount of DNA) I would precipitate the eluate with like 75 ul of 100% ethanol, leave overnight at -20 and then centrifuge at 12 000g/10 min/4 degrees and discard supernatant, briefly dry and then dissolve in what amount of water or buffer you want (but note you probably won't see any pelet, but unless it's a really low DNA concentration, it would be there, I use this protocol for desalting PCR products after restriction).

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#6 harry348

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Posted 05 June 2013 - 10:43 PM

thanks Trof




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