DNA extraction from the gel - low yield - (Oct/30/2013 )

Hello everyone,

I have a plasmid with 8kb insert and I need to purify this insert in large quantities (up to 5ug) for a subsequent transformation of fungi.

So I digested 3ug of the plasmid, run a gel, cut a gel fragment with the insert and extracted DNA with Qiagen kit - but the yield was very low (0-5ng/ul)

For the next extraction I used warm elution buffer, increased elution time (10 min instead 1 min) and also used lower concentration of agarose (0.8%) - again, yield was about 25%

Then I tried an old-fashioned method - centrifuged piece of agarose, collected supernatant and precipitated DNA with ethanol. DNA concentration was 100ng/ul in 60ul which is 6ug of DNA - more, than was in restriction reaction. Also, according to the Nanodrop measurements, DNA was not pure.

What I want to do next - pool all samples together, concentrate them with SpeedVac and do phenol/chloroform purification.

Can anyone suggests what else can be done to increase the yield? 

Have you considered using PCR to prepare your insert. You could avoid the gel entirely. Also, do you really need to remove the other part of your vector? Perhaps you can cut, purify, and use it directly. I'll do most anything to avoid running preparative gels.

Thanks, phage434!

I'll ask my supervisor if I can do the transformation without removing the vector backbone. PCR will be a step back because I would need primers and polymerase for the long PCR.

Gel extraction is OK for small fragments but not as good for larger DNA's.