In which concentration and volume (microliters) DMSO 5% or 1-10% I can used to optimize the PCR for specific product to avoid nonspecific product? I am facing a problem that I have use 1-10% and than only 5% and 10% DMSO in PCR. but result is smearing of product or no product. guide me how can I optimize PCR.
You do not know in advance because the region/s of homology can be very varied and longer GC tracts ,for instance, may need more dmso. The best way to find out what concentration to use is to run the same sample at 1%, 2%, 3%.....up to 10% dmso in the reaction mixture. If this is not working then you need to check that your dna sample is good quality ( run a different primer set) and that your primers are capable of working ( run 4 different samples). If the primers are old or inherited from another researcher then order more primer and dissolve them in TE not water. Check the quality ( od260/280 is about 1.8) and run a pcr with samples of 10ng, 20 ng,59 ng and 100 ng. If you have pcr inhibitors in your dna sample the weaker dna samples may work but the concentrated ones will fail. You can also try a MG titration. Amplify 1 sample with 1mM,2mM and 3mM Mg because sometimes the dna of the sample or the large amount of dna removes MG and the enzyme needs at least 1nM magnesium to work. Run a no template control in all your experiments in case you have pcr product contamination. If you get a smear in the NTC then you have contamination and over amplification and that will be a problem
Hi Madeeha
in addition to Paul Rutland 's as ever best answers, you should also take some time to test in silico your PCR (if it doesn't fit, it will never work).
go to UCSC (http://genome.ucsc.edu/cgi-bin/hgPcr) or at NCBI (https://www.ncbi.nlm.nih.gov/tools/primer-blast/index.cgi?LINK_LOC=BlastHome) just to be sure.
all the best
fred
- Badges
- Science method