enzymatic digestion - (Apr/05/2002 )
hi! I'm trying to digest a RTPCR product with 2 restriction enzyme but I'don't have any good results : on 2% agarose gel, I can see some expected bands but not all of this (2100,647,347,268,247,180 pdb). Furthemore the fluorescence is weak despite of a good coloration in BE and of use of a great amount of RTPCR product. Before the digestion, I purify the product with a Quiakit pcr purification kit. The time of incubation is 2h30 (37°C).
Do you have a solution for this problem?
Are you sure of your kit? Is it really a good one, or is it really necessayr, because i have the feeling that kit catches a part of your sample.
Have you tried using BSE in the RE buffer? I know that it sometimes helps if the enzymes aren't cutting. The other thing to try is spermidine, but I would try the BSE first. You may also want to try incubating overnight to make sure that the cDNA is cutting fully. Good luck!
Seems I have cow diseases on the mind. I should have written BSA (bovine serum albumin) not BSE. Sorry about the confusion.