Can we purify Miniprep DNA and use for transfection? - (Oct/28/2005 )
I am having trouble with Qiagen Midi kit and getting very low yields of plasmid. The DNA that I got was only 600 ng/50ml culture. I am able to get high yields with Miniprep. protocol approx. 20ug/50 ml. I wanted to know if there is some way I can purify my miniprep with the Qiagen columns.
if you mean that you extract from miniprep by columns, i think it's a good quality of plasmid. But i'm wondering if it also removes endotoxins...
but 600ng for 50ml culture sounds quite reared. I would advice you to double the amounts of solutions recommended...
I have had this work for me, but I have to say, I never have as high a transfection efficiency with DNA isolated from minipreps. If I EtOH ppt. and concentrate several minipreps, it usually works better. Anyone else seen this?
I always EtOH precipitate my minipreps before transfecting, just to reduce the volume to actually transfect.
and indeed, as Fred says, a way to increase your yield is to increase the volume of lysis buffer (and afterwards increase the neutralisation buffer also). I just increase with 2-3 ml and it really helps me. Another way might be to use a richer medium than LB, like TB or so.