Oligo purification - (Jul/08/2005 )
I am performing primer purification. I am using PAGE to isolate them and then I will cut the band and squize it. According to Maniatis' protocols I should run a huge gel at 1500V but the plates crushed even at 1000V. At what voltage do you do it?
OMG, I am so sorry you have to PAGE purify your own primers. I've checked primer integrity on a gel before, but never purified.
Why do you need such high voltage anyway? This isn't a sequencing gel.
Run the primers on a denaturing acrylamide gel at a reasonable voltage, cut out the band and do freeze and squeeze like Maniatis says.
I am still in doubt: which is a reasonable voltage?
and do you know a kit for oligo purification?
After break two plates, I did it at 500 at it functions ok. I runned it in the protean 2 (the big one) from biorad.
As I told you I was able to succesfully run the PAGE gel for my impure primers. I enveloped the gel in saran wrap and I put it over a silica gel plate. Then I iluminated with UV ligth and the position of the primer bands was visualized by a shadow (cause the DNA absorbs the UV ligth). I recommend this procedure to investigate if your primers are well purified.
Now that I got the band slice I am not sure how to elute the primers. I want to ask some ideas.