Which gel concentration - (Jan/29/2011 )

I'm trying to separate human allelic bands by gel electrophoresis, and these fragments are between 150-400 bp long. What concentration agarose gel would I need to resolve these bands? I am making a comparison between different peoples DNA, so I need to seperate the bands to an extent where I can differentiate between the different people.

I'd try frist a 1.5-2% Gel and lithium borate buffer (instead of TAE/TBE), a sufficient long gel is necessary too. But I guess you'll never reach the resolution of a sequencer i.e. a long polyacrylamid gel.

hobglobin on Sat Jan 29 19:33:23 2011 said:

Hi hobglobin,

Thanks for your response. I'm yet to try the lithium borate buffer. Have tried TAE and sodium boric acid (as seen in Brody and Kern, 2004), but lithium boric buffer is my next step

What length gel would you recommend? Do you think a 12 cm gel will do it, or would a 20 cm be better?

Baileys on Sat Jan 29 20:12:59 2011 said:

The longer, the better I'd say.

Baileys on Sat Jan 29 20:12:59 2011 said:

Would just like to add that TBE buffer is better at resolving smaller fragments. A 2.5% gel in TBE separates bands 20-30 bp apart for me after 50mins of electrophoresis.

Hope that helps!!!