PCR, labelled vs unlabelled primers - (Aug/08/2006 )
Hi everyone!
Ive been running some PCRs. I run multiplex PCR with three loci per reaction. Unlabelled primers yield products, while identical but labelled primers for corresponding loci give no result.
This ccould of course be due to contamination of the labelled primers etc, but are there other factors that could contribute to this phenomenon?
Cheers from Chimp
-Chimp-
What labels are you using, and where are they?
-swanny-
Check the sequence of the labels. Could they be STOP codes? (it's been done before). Might be worth sequencing the labelled primers and checking you have what you think you have.
-paraboxa-
QUOTE (paraboxa @ Aug 14 2006, 02:14 AM)
Check the sequence of the labels. Could they be STOP codes? (it's been done before). Might be worth sequencing the labelled primers and checking you have what you think you have.
Im using NED dye at 5' of the primer.
The sequences are the same for labelled and unlabelled primers.
-Chimp-
the only thing I can suggest, is perhaps somehow the label is interfering with binding
have you tried an alternate label?
-aimikins-