QuiaQuick Spin column - (Apr/10/2008 )

Hey all,

I amplified a DNA fragment by PCR and wanted to purify it before restriction digestion. I used Quiagen PCR purification kit (with qiaquick spin columns). I started with 30 microlitre reaction mixture and finally eluted with 30 micro litre Elution buffer. I measured the concentration on Nano drop and it showed 20 ng/ micro lit. That means i have only around 600ng.

Do you have any experience with this kit? If so how good is your recovery. I am particularly worried since after restriction digestion, I have once more purify the fragment to get rid of enzymes before ligation. I dont want to lose more DNA.

Also can anyone suggest me if it is better to go for a Gel elution kit or direct PCR purification kit.

You can typically improve elution from Qiaquik columns by heating the elution buffer to 50C and letting it sit for a few minutes on the column. Also, a two-step elution works better than a one step elution. Elute with 30 ul first, and then with a second 20 ul. You probably did not get 30 ul out of your elution, since the column holds up a significant volume. Also, at the start, you can flow the pcr reaction + buffer PB through the column twice, which improves binding efficiency.

If you design your reactions with restriction enzymes which can be heat killed, then you need not purify the restriction digest prior to ligation. In general, the fewer purification steps you use, the better your yields.