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ethanol precipitation plasmid DNA


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#1 bioke

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Posted 26 June 2014 - 10:31 AM

Dear all,

 

Should I add sodium acetate when doing an ethanol precipitation of plasmid DNA after I digested it ?

 

The salt does not seem needed because the buffer itself also contains salts.

 

Or is it Always needed to add the sodium acetate?



#2 phage434

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Posted 26 June 2014 - 11:40 AM

The sodium acetate adds 300 mM salt -- a dilution of 10x from the initial 3M stock concentration. This is higher than most digestion buffers, but you may not need that much salt to do a precipitation. Do you want it to work, or do you want to do the experiment?



#3 bioke

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Posted 26 June 2014 - 12:30 PM

I am not really sure what you mean by: do I want it to work or do I want  to do the experiment?

 

I want to collect the DNA between digests thats all (and just for future work). I am wondering if the sodium acetate is really needed. Nobody in the lab adds the sodium acetate while pretty much all protocols I can find mention this sodium acetate so I wonder if its really needed to add it.

 

The sodium acetate adds 300 mM salt -- a dilution of 10x from the initial 3M stock concentration. This is higher than most digestion buffers, but you may not need that much salt to do a precipitation. Do you want it to work, or do you want to do the experiment?

 



#4 phage434

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Posted 26 June 2014 - 04:23 PM

What I meant is that you can add sodium acetate, precipitate your DNA, and move on with your protocol, or you can try a different technique, which may or may not work, and which will be an experiment. Depends on what your goals are, doesn't it?






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