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Ethanol precipitation without using salts - (Dec/29/2010 )

I didn't put any salts to precipitate my purified plasmid in ice-cold abs EtOH because my supplier of miniprep said i don't need to because the salt is for purification. But then i searched online for that and found out that the salt makes DNA less hydrophilic and suppose it has nothing to do with purification. Besides that, i put 950ul of abs EtOH for my 40ul plasmid. Would the DNA denature or anything?

-hianghao-

I think it depends what you will do with your plasmid later 'cause salt can interfere with later steps. I am using the ice-cold EtoH+centrifugation for precipitation of the plasmid. 

Also, additional advice: if you are doing "home-made mini prep" (not the one with commercial kit and silica membrane), be sure to dry the EtOH before you add TE-buffer(or water) for storing the plasmid, so it would not interfere as well..

Hope I helped a bit.

Cheers :)

-lab_microbe-

The final neutralized supernatant in a typical commercial miniprep has more than enough salt to allow precipitation with just adding alcohol. After the 70% alcohol washes, however, much of the salt is removed (the purpose of the washes). When the DNA is redissolved in TE or water, it will not precipitate again without the addition of both salt and alcohol. So, the answer to your question depends on where the DNA solution you are trying to precipitate comes from. If it is the supernatant from spinning down neutralized cell lysate, then you don't need to add salt. If it comes from (for example) elution of a column with water or TE, then you do need to add salt.

-phage434-

lab_microbe on Thu Dec 30 09:16:40 2010 said:


I think it depends what you will do with your plasmid later 'cause salt can interfere with later steps. I am using the ice-cold EtoH+centrifugation for precipitation of the plasmid. 

I see... I want to sequence the insert.

phage434 on Thu Dec 30 12:50:31 2010 said:


If it is the supernatant from spinning down neutralized cell lysate, then you don't need to add salt. If it comes from (for example) elution of a column with water or TE, then you do need to add salt.


I am using plasmid eluted with TE from column. And i didn't add salts as the miniprep supplier told me i don't have to :( Can i add the salts after abs EtOH is added? Or is there anyway to retrieve my plasmids from abs EtOh?

I read in some online articles that say vortex when wash DNA with EtOH, but some ppl say vortex only if RNA. Should i vortex or not? IF DNA can break in vortex, why can't RNA?

-hianghao-

Yes, you can add salt after the ethanol and precipitate DNA. After precipitation, you should wash the pellet with 70% ethanol to remove the salt from the pellet, then dry for 15 minutes or so with the tube open (until the ethanol smell is gone) and then redissolve in TE.

Vortexing plasmid DNA makes little difference, since it is physically small. Vortexing genomic DNA, which can be centimeters long if stretched out, will break up the DNA. RNA is usually quite small, so there is little issue with vortexing RNA.

-phage434-