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?