PCR problem: DISTINCT bands far shorter than target!
Posted 26 February 2005 - 01:26 PM
Here's my rxn. conditions:
1ul template (cDNA lib)
10ul NEB thermpol buffer
10ul of each primer @5uM
3 ul dNTP's @ 10 mM
1ul Vent pol.
water to 100ul total volume
I am new to PCR and any help would be awesome!!!
Posted 26 February 2005 - 04:09 PM
I had that problem also with a big (~2kb) product and addition of 5% DMSO solved the problem. You could test several DMSO concentrations as you have to add the less needed as DMSO increases the rate of mistakes !
Good luck !
Edited by Panoramix, 26 February 2005 - 04:12 PM.
Posted 27 February 2005 - 08:59 AM
I do 38 cycles at: 94.0 (1min), 55.5 (1min), 72.0 (2min)
Posted 27 February 2005 - 06:54 PM
I was just wondering, does the gene sequence length include introns because if that is the case, if you are amplifying from a cDNA library, ie: DNA from mRNA transcripts which would be devoid of introns and hence the shorter product from expected gene length.
If you are getting two products from the one primer pair this would possibly mean alturnative splice transcripts from the same gene.
Hope this helps