Problems after gel purification of plasmids - Restrictases and ligase do not work on gel purified DNA (Jul/25/2007 )
I am wondering whether anybody encountered a similar problem.
I cut a plasmid with SalI, then ran though Qiagen gel purification system and cut with EcoRI. EcoRI digestion did not work after the plasmid had been run thru the column; however, if I add the enzyme directly without prior DNA column purification, it works fine.
Similarly, I tried to ligate a gel-purified cut vector and an insert (1:3 ratio, 150ng total DNA) with NEB T4 ligase (0.5 U in 10ul) at 16C overnight. When I ran the ligation mixture on a gel, I saw that it did not work. I suspect it is because of the gel purification columns.
Has anybody experienced similar problems? My lab mates say they have never seen this. Help...
PS: I tried eluting DNA with either water or Qiagen elution buffer; it elutes fine in both cases, but enzymes do not work afterwards.
Make sure you are doing the dry spin to remove all of the PE wash buffer from the column. It sounds as if excess ethanol from the wash is getting into your sample.
Dry spin at maximum speed was immediately suggested by my lab mate. I tried it, but the outcome did not change. Must be something else... Any more suggestions, please?