Agarose gel concentration for small and similar pcr pdts - (Jun/27/2007 )

Hi,

I'm hoping to separate PCR products of ~110bp and ~100bp. I've used 2% and couldn't see the difference. As I've never try separating such similar size of pcr pdt before, what concentration of agarose gel should I use? Your help is much appreciated!!

ketup

I'd recommend using an acrylamide gel for better resolution. (After all, this is what they use for DNA sequencing...)

Ginger

This is not really a easy job..

either your hude acrylamide gel .. but even its not easy..

but you try beads..

like .. you can make two anchor oligos (specific to your samples) and stick them to beads seperately..
And then if mix your both products.. specific sample with bing to specific bead.. n you will get highly pure sample..

Thank you for all your suggestions!!

ketup

Can use metaphor 3-4% (is a little hard to make, it burn easily and must pour very hot), but it separates really small fragments.

3-4%? I think it is quite hard to make those concentration. I still agree with er.. ginger spice to use PAGE gel. Better resolution.

Yes is a little bit tricky, but can be done. Is better 3%, hidrate the agarose in cold buffer before melting. Melt in the microwave at 75% power and pour a little bit hotter than the normal agarose (don't pour directly from micro to gel caster or it will crack). After it polymerize put in a plastic bag at 4C until need. This type of gel have a better resolution when let it cold for at least 30 min before run. Run the gel as normal.