Does residual ethanol in DNA inhibit PCR? - ? (May/31/2005 )
I am using a new DNA isolation procedure which is based on salting out. I'm obtaining the genomic DNA from serum and taking out the DNA by pipette which is precipitated using ethanol. small amounts of ethanol is also transferred to the TE. I obtained PCR product in some samples but not in others. Is there anyone using such a method and facing such a problem? I thought that I can do centrifugation so that to obtain DNA in pellet and get rid of ethanol completely. Am I right?
as I understand your technique, you directly take the DNA from the EtOH-precipitation
to the TE-buffer, then you use this as template for a PCR. The EtOH-carry over
seems to me quite high with this method.
I'm also using a salting out method to isolate bacterial genomic DNA (NaCl +
Chloroform-extraction), I precipitate the DNA with isopropanole, "fish" it with
a glas hook made out of a pasteur pipette and transfer it into a new 2 ml tube.
I pellet it and remove residual iPrOH. Then I wash it with 70% EtOH and dry it
for a few minutes under vacuum. After that I resolve it in Tris-buffer overnight
at +4°C, because it's very much DNA and the pellet needs some time to get
But the PCR and Restriction digests work very nice with this method.
So, I would suggest to pellet your DNA and remove residual EtOH, if possible
dry it under vacuum, 3 min should be enough, then resolve it in TE.