Ethanol precipitation - do you mix after adding salt? - (May/20/2005 )
when you do ethanol precipitation, does it make any difference if you mix well after you add salt (e.g., sodium acetate) or not ?
I only mix after having added bot salt and ethanol. It is very important to mix well after that. I knew this because sometimes if my samples were not well mixed, the bottom part got frozen at -20C.
I agree with Green ... mix very well after the addition of both the salt and ethanol!
I also think it is important. In some library manual, It is refered that you can just add 2 volume enthnol(without salt ) and mix well, put it in RT for 2 minutes and then centrifuge at 4C. I did this and got the product. compare with the method adding salt, Which one is better? Give me your comments. Thanks.
It depends on how moch salt is in your sample to begin with. The idea of adding the NaAcO is to convert the DNA into a "giant" salt molecule which will then precipitate in EtOH. The amounts of salt added are huge excess and you can probably use less or none if your original buffer has a high enough ion content. I find that using the standard 1/10 volume 3M NaOAc then two vol EtOH works every time. If you want maximum yield I would mix well after all steps and preipitate at -80C or even on dry ice. If you have lots of DNA and you don't care if you lose some then just add EtOH at room temp, mix and spin.