pcr problems - pcr to solve (Dec/21/2007 )
hi everyone..i am trying to amplify the 2.3 kb gene and the source is cDNA. Before that i checked the quality of the cDNA by using the four primers. Lets say 1 2 3 4 primers. 1 and 2 will amplify 500bp and other 3 and 4 will amplify 702bp. i got the result and quality is good.....but i try to amplify the whole gene with the primer pairs 1 and 4 , but not succeded..does anyone have the solution please...
some ossible problems:
* inadequate extension time (2.5 minutes for most enzymes and a 2.3 Kb region)
* primers 1 & 4 form primer-dimers (test with IDT tools, www.idtdna.com)
* you're really amplifying gDNA not cDNA and there is an intron between 2 & 3
* hairpin or high GC region between 2 & 3 (add 5% betaine)
* very low GC region between 2 & 3 (lower extension temperature to 65)
go over the sequence of the gene to identify any GC rich regions. If so, you could use enzymes which are better for GC rich amplification or use DMSO with your enzyme.