which step in the pcr cleanup process could harm the cohesive ends - (Feb/20/2012 )
Hi dear guys,
Thank you in advance.
rihgt now,i am trying to ligate the plasmid with two virus genes.After using restriction enzymes to cut them,i use the promega wizard pcr cleanup system to purify them.I just wanna know if any step during the process of the purification could harm the cohesive ends of the DNA,according to your experience?
The ends are readily accessible to DNAse enzymes, so it is important to inhibit them by removal of Mg++, a required co-factor. I'd recommend eluting in TE and storing your DNA in TE. Unless your reactions are dominated by a high volume of your DNA solution (which means you may have other problems in any case) the EDTA in TE is so low in concentration that it has a minor effect on final buffer Mg++ concentrations.