Degradation of PCR product during purfication - Purification of PCR product for cloning (Apr/12/2006 )
I have some problems while purification of PCR product; I tried purification by two methods
1) By QIAGEN - QIAquick GEL EXTRACTION KIT"
2) By phenol chloroform method in which I cut the desired band after addition of equilibrated
Phenol (pH 8) I incubate it at 65°C for 20 min. than I transfer it to -70°C for 30 min than after centrifugation I take soup than chloroform washing than I use to add 100% ethanol double the volt. of upper layer (I got after chloroform washing ) and in this I add sodium acetate ( 3M pH 5.2 ) 1/10 of total vol. than 1/2 hr in -70 c followed by centrifugation 14000rpm than washing by 70% ethanol ,than air dry followed by addition of autoclaved dist. water.
as I tried both the methods but when I check my product on agarose gel I got 2 bands ,also product with which I start is single band without smearing.....
Please suggest me something to trouble shot this....
Waiting Ur reply......
thanks a lot
How big is the difference in size between the two bands? Maybe on your original gel, you have loaded so much DNA that you cannot distinguish between the two sizes?
are you using UV to check your bands? If you leave products under UV too long it degrades the DNA which may be why your seeing two bands? why are you gel purifying anyway if you've got a single band? Try qiagen PCR purification kit instead.