Primer design - GC clamp
Posted 06 August 2009 - 07:49 AM
Posted 06 August 2009 - 08:44 AM
Hope this helps!
here is a Link
Posted 31 August 2009 - 10:47 PM
As far as I know, having a high GC content facilitates tight binding of the oligo to the template but also increases the possibility of mispriming.
May I know, which software are you using to check your primers? I use NetPrimer from Primer Biosoft: http://www.premierbi...imer/index.html
Posted 01 September 2009 - 05:30 PM
Posted 02 September 2009 - 08:49 AM
Hope this helps.
Posted 18 February 2010 - 07:38 AM
This question about the GC clamp on 3' ends of primers interests me a lot.
Right now, I'm using Primer 3 to pick primers from a sequence and in the GC clamp parameter field, I've chosen GC clamp equal to 0, 1 or 2 and picked primers for each situation (everything else equal). The resulting reverse primers were different for each and only the forward primer resulting from a situation of a GC clamp = 0 was different from the others.
I've had some problems trying to amplify this locus with the original primers designed for it. They're supposed to work in a conserved region (an exon), but the amplifications I got were never consistent when it comes to the amplified band intensity. So I decided to try and pick other primers more inside the exons flanking my target sequence, in hopes of having working primers that might not be subjected to any putative mutation.
So, I wonder if the primers picked with GC clamp = 0 will force a less specific amplification, but on the other had will increase the changes of yielding any product at all, specially to proceed to sequencing afterwards?
What are your opinions on this?
Listening to: Interpol - Say Hello To The Angels
Posted 18 February 2010 - 11:43 AM