PCR amplicon size and contamination - (Sep/27/2005 )
Hi. Quick question, is it easier to pick up/amplify contamination when using primers that amplify a small region? E.g. when I use primers for large product (1500bp), I get no visible contam, but when using primers for smaller regions (product expected = 200bp), then I always find I have contam problems (I've changed primer and all other stocks a few times) - The primer concentrations are the same for both sets of reactions.
I have even done the pcr with both sets of primers at the same time, used the same reagents and thermocycler and ran on the same gel, and the negative with sterile water as template and the "large product" master mix was clean, while the negative with sterile water as template and the "small product" master mix had a band the same size as expected product! So I was just wondering if this is so, or if anyone else has had similar problems, or if it's just me!!!
Thanks in advance for any replies!
May not be contamination. It could be dimers or non-specific amplification.
But non-specific amplification in the negative control? There shouldn't be right, considering there's no template added? And how can I be sure if it's primer dimer? Thanks!
you can perform a melting-curve analysis
you can make a primer-dimer deliberately and run it on a gel, and compare (just set it up like an oligo annealing reaction. if you are going to get primer dimers, they will show up without any effort on your part except an increased temp)
You could also sequence the pcr product.