Primer Dimer in NTC Only?
Posted 12 September 2009 - 09:04 AM
Background: I've been starting up qPCR in our lab, we have an ABI 7500 fast system and I'm using the Fast SYBR Green Master Mix from ABI. I'm working on determining primer efficiencies for primer sets to be used to analyze chromatin immunoprecipitation data. The amplicon size for the primers range from 250-350 bps and are tiled along the presumed promoter region of our gene of interest.
Issue: I've examined efficiencies for some of my primer sets and they come out quite good (around 95%), however, I seem to get amplification in my NTC wells that appear to be primer dimers. In my template wells (mouse gDNA) at 1:1. 1:10 and 1:100 dilutions I get very nice amplification, clean melt curves (no shoulder) and a nice solid band on my gel. However, in my NTC wells (typically 2/3) I also get amplification that is not my product and has a lower Tm. Upon running on a gel it appears as a diffuse band below 100bps (looks like a primer dimer, smells like a primer dimer...).
Question: Is this an issue for experimental analysis? Can something like this be solved simply by playing with primer concentrations? Or does it require primer re-design? Would switching master mixes potentially help (e.g, to Power SYBR Green)? I'm a bit limited in my genome region as I need to stick around my presumed promoter region for the ChIP.
Thanks for considering my question.
Posted 13 September 2009 - 01:36 PM
Posted 17 September 2009 - 04:27 PM
Posted 18 September 2009 - 12:42 AM
Your suggestion sounds good though, in my primer sets in which I only seem to have a small primer-dimer problem I'll try playing with the annealing temperature. I've just been using a 2-step PCR as recommended by ABI for their master mix (95C and 60C) and hoping that will give me the best results.
I agree with Jah, you need to play with annealing temperature.
And you can give me your primers, I'll check it in Vector NTI.
Posted 20 September 2009 - 01:09 PM
All the figures below represent the derivative output from the melt curve for NTC and mouse gDNA samples.
Primer 8(1) seems to produce a very clear primer dimer at a Tm immediately prior to the expected amplicon.
Primer 58(1) has a broad "shoulder" prior to the expected amplicon.
Interestingly, in both sets there does not seem to be any primer dimer amplification in the NTCs. On the gel for primer 8(1) there is clearly only one band of the appropriate size. My gel with 58(1) didn't come out well enough for me to get a good look at it, but it basically looks good in terms of specificity.
Would increasing the annealing temperature work better for one particular melt curve outcome versus another?