I am using multiplex PCR of genomic DNA in groundnut. One is an allele specific primer and the other is a universal primer.
I suggest to minimize the conc. of the primers to F: 0.3, R1: 0.2 and R2: 0.1.
Also i think lowering the MgCl2 conc. to 1.5 can be beneficial. Please let me know the results. We have used this approach for setting up our multiplex for 7 target genes and it worked.
Dear Sahil, can let me know detailed reaction condition (MgCl2, Primers, Taq and dNTP conc.)?
Dear Reza Raoofin, MgCl2 - 2 mM, dNTPs-0.2 mM, Primers- forward- 0.5 µM, Reverse 1 - 0.4 µM, reverse 2- 0.1 µM, Taq polymerase- 1 U, Template DNA - 40 ng.
Dear Reza Raoofin, I run the PCR products in a 2% agarose gel.
Do you have non specific bands, smear along the wells you have run the samples or excess (unused) primers?
I suggest to minimize the conc. of the primers to F: 0.3, R1: 0.2 and R2: 0.1.
Also i think lowering the MgCl2 conc. to 1.5 can be beneficial. Please let me know the results. We have used this approach for setting up our multiplex for 7 target genes and it worked.
You can minimize your primers and dntps, check your DNA quality (might be fragmented) and you can try to use thiourea in your running buffer (50µM).
Dear Reza Raoofian, i am not getting any non -specific bands, only a smear is there. i have attached the gel image. the Tm of the primers is 66 degrees. i am loading 5 microliters sample per well.
TO dear Vijay Chitnis and Michael Granitsiotis, the quality of DNA is better.
O.D. = 1.83, Concentration = 19.80 ng/µl
When I say quality, I mean, run it on a gel or even better in a bioanalyzer chip before you do your PCR...You should see if it is fragmented before you use it. Good quality DNA normally is showing a smear above 5 kbp. If you have a smear bellow 1kbp, try to purify it again with a column purification kit (assuming that you have enough DNA to do so...)
Dear Michael , i have run the gel on 1% agarose. it is not showing any fragmentation.
Dear Sahil, i have attempted to download the image 4 times and every time the file seem to be corrupted. Can send it via mail ([email protected]) ?
Dear Sahil,
to me the gel looks as if the smear is from your master mix maybe? What is in left bottom lane (left from the DNA ladder)? I am not so familiar with Multiplex PCR - however the lanes with no clear band look as if a primer is missing/not binding resulting in linear amplification. Did you try the primers also for linear amplification - decrease annealing temperature to 50 or 55 degrees? This might help you if one primer is the problem.
Good luck & keep us updated ;-).
I think Dennig is right, check the annealing temperature and hope it will work for you.
In Multiplex PCR, DNA quality is more important aspect.
is smearing effect is present in all band? than optimize the PCR profile and reagent incorporated or if in few band than Check the DNA Quality.
Dear Alexander Dennig, the left bottom lane is a negative control without the DNA template. the tm of the primer is 64 and 63. i am using a touch down PCR from 64 to 60 degrees. i am getting the desired band, just the problem is about the smear.
Dear Reza, still i am getting the smear, though the intensity of my band has increased.
Dear Yanuar, after decreasing the cycles from 35 to 30, the bands are getting fainter. i need dark bands with no smear...i am trying a lot of modifications still i am not getting the right match.
What template you are using, try to reduce the template and see what happens
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