purification of PCR product

[ranju]

hi guys,
I am trying to purify 120bp pcr product by running it on denaturing polyacrylamide gels. I was succesful once. But now I am not able to repeat it. can u suggest me a better way of purification.
thanks

[kmunson779]

It depends on what you're trying to do.

I would use a non-denaturing polyacrylamide gel for a 120 bp PCR product, if I needed to gel-purify. You could try 2% agarose and see if you get sufficient separation to cut the proper band. If your PCR looks clean (only your 120 bp product) you could use a column-based PCR cleanup kit rather than gel purifying.

What do you mean by you couldn't repeat it? Couldn't see your band, get DNA out of the purification process, or get downstream processes to work?

[oBWhat]

Hey Ranju,


there are many great kits for PCR product purification such as Qiagen.

Ive had many problems with small DNAs such as the one you are using (120 bp)

The problem is even with kits, the minimum is around 100 so much of your DNA is being lost.

Either make like 10-20 PCR reactions and load then into many lanes, and extract several lanes (4-6) into ONE PCR purification column

thats what i did...