ppt DNA directly from a digestion/ pcr - (Oct/27/2006 )
how advisable is it to precipitate DNA directly out of a restriction digest or a PCR (instead of gel purifying it) and thereafter carrying out a ligation on the precipitated, freshly reconstituted DNA?
thanks for your help!
i would suggest running it on gel if it has been digested to get rid of the ends which might other wise interfere during ligation.
so I wouldnt advise to ppt DNA after digestion and use it for ligation.
for a PCR product that has just come off the thermo cyclers, I would strongly recommend you either gel purify the DNA or clean it using a column binding kit. EtOH percipitation will percipitate dNTP along with the PCR product. Thermal stable polymerase is resistent to such treatment, thus there will be some low level activity from the polymerase during the ligation.
As for the digest, do so only if your vector does not have a restriction site used to cleave your insert. There will be some low level activity from the restriction enzyme as modern recombinant restriction enzyme is fairly robust. It is best to either phenol chloroform the digest before ligation or do a gel purification.