Gel or column purify - (Jan/17/2008 )
I am wanting to remove CIP from the ends of my vector before proceeding with my ligation. I am wondering if gel purification would give a cleaner result than spin-column purification? What are the guidelines regarding when to use spin-column and when to gel purify (if I have only one size of DNA in my tube)?
heat inactivate it and do a gel purification.
For cloning, I like to gel purify. The rationale is that if you are digesting a piece of DNA, then you should get two pieces as a result. In the case of a PCR product, this will be your gene of interest and a tiny piece off each end. If you spin purify, both of these pieces are likely to be eluted. This means when you go to ligate, it is theoretically possible to simply reattach the ends to your PCR product. In gel purification, these two fragments are separated and you only extract the gene of interest (the smaller piece travels farther in the gel).
Does that make sense?
I usually just add CIAP to my vector digestion mixture during the last 20 minutes or so, then gel extract both fragments and ligate.
This depends on the length ofthe ends you cut of. Most spin colum-kits are on the market for the removal of primers (and taq) after PCR reactions, so if you only cut near the ends of your PCR product, you should also loose the cut pieces.
This depends on the length ofthe ends you cut of. Most spin colum-kits are on the market for the removal of primers (and taq) after PCR reactions, so if you only cut near the ends of your PCR product, you should also loose the cut pieces.
Definitely a good point. I believe the manuals for spin-column kits should specify the cutoff value.