One of the main reasons why primer dimers occur is because of DNA sequence complementarity within and between primers. Because of this complementarity the primers simply come together efficiently, which causes primer dimers.
The best way to get rid of primer dimers is to design the primers in such a way that there is no complementarity within and between primers. One good approach is to design 3 different primer pairs and chose the pair that works the best (that generates no primer dimers).
When it comes to primer dimers, regular PCR and qPCR are the same. All you need to do is design the assay in such a way that primer dimers do not form. Every time I designed a new qPCR assay I always tried at least 2, and many times 3, different primer pairs to see which one worked best (and got rid of any assays that formed primer dimers).
I have been trying to do PCR (conventional )to amplify a particular housekeeping gene loci, but I am not able to get any bands.
I tried various combinations of primer ( 0.2- 1 um) and template (50ng/ul ; 1- 5 ul) concentrations but to no avail. Also i tried adding extra mgcl2. I am getting bands in a few samples and that too not consistently. Also there is the added problem of primer dimers (even when a band is obtained ).
How do i avoid getting primer dimers and increase the efficiency of amplification ? please help!
You need to analyse carefully your results and decide the best action to take as first step.
I will do the following steps to find a solution.
1. If the band obtained is the expected band(should be confirmed by sequencing or at least by restriction digestion), the associated primer dimer issue could be resolved by reducing the primer concentration for final PCR. Prepare a higher dilution of the working primer and use for PCR.
2. If you are not able to get rid of the primer dimers and it does not interfere with downstream applications, I will not worry about it as far as my product is the correct bp band.If necessary, I will do gel elution step to get rid of the band.
3. If the band is not the expected PCR product bp, I will re-design the primer pair for the PCR.
4. In consistent bands could be due multiple reasons needing thorough evaluation. The reason could depend on sample, micropipettes, instrument used for PCR, quality of water used for PCR set up and so on. It is not a simple matter of adding extra MgCl2.You should also try PCR without adding any MgCl2 at all.
5. If you need more guidance, please upload a gel picture of 2% agarose electrophoresed fragments.
Hope this help.