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Ethanol precipitation - bad step


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3 replies to this topic

#1 Katuss

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Posted 22 May 2012 - 11:07 PM

Hello, could you help me please ? I want to ask if it´s a problem when I added ethanol to my plasmid and put it in to -80 dedrees of Celsia overnight. I made a mistake and don´t know if I can continue. Thanks.

#2 phage434

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Posted 23 May 2012 - 04:10 AM

It's almost certainly recoverable. What are you doing with it next? You could purify the plasmid by essentially doing an ethanol precipitation. Add 1/10 volume of 5M NaCl, 2.5 volumes ethanol, spin down hard and look for the precipitate. Wash with 70% ethanol, and finally redissolve in TE. If you started with salts in your initial solution, or if you added sufficient ethanol, you could omit some of these steps.

#3 BMF

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Posted 24 May 2012 - 03:50 PM

DNA in alcohol is safe always. Alcohol is a temporary inhibitor for DNases.
For precipitation you can add Alcohol and then keep it in RT, -20, -80 or whatever you want.

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Babak Memari

#4 Katuss

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Posted 26 May 2012 - 02:04 AM

When I put out the DNA I put it into -20 for one hour and then centrifuged for 30 min and washed with 70% ethanol and redissolved in H2O (because of the next using in cells) but the concentration was small again, about 40 ng/ul ...so I think when it was in -80 it didn´t work, DNA is not damaged but the precipitation doesn´t work in these conditions.




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