3kB PCR Cycling Conditions?//
tedm at darkwing.uoregon.edu
Fri Jul 21 22:28:50 EST 1995
In article <3ucuiv$imf at news.ycc.yale.edu>, burbly at minerva.cis.yale.edu
(Marc A. Borbely) wrote:
> Hello. I'm trying to amplify a 3kB fragment from mouse genomic DNA. I've
> been using Taq polymerase with a 3 minute extension time, and 20 seconds
> annealing, and 20 seconds denaturing. I'm getting no product. Does anyone
> have better cycling conditions I should try?
> - Marc Borbely
What is "no product" ? Is there a nonspecific smear? or nothing? An
internal control is sometimes useful, like the the beta globin gene for
human genomic PCR. Your times are at least in the ballpark for a 3kB
extension from Taq, no Vent additions or other "long PCR" stuff should be
necessary. Check the primer length and the annealing temp, refer to the Tm
discussion in this group, and make sure the annealing temp isn't too high.
Also make sure all the amounts are correct (dNTP's , primers, etc) Lastly
triple check the primer sequence and the orientation, it has to be just
so! Are you going enough cycles to see product from the copies initially
present? etc etc. Best to show your stuff to a person who has trouble shot
PCR (ie a person who has done 5 or more PCR's)
Good Luck, Feel free to Email with specifics if others can't help...
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