Ethanol precipitation - bad step - (May/23/2012 )
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.
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.
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.
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.