I have done bisulphite treatment manualy as well as with kit from zymo and then methylation specific PCR. but , I am getting primer dimer in 2 out of 3 genes and no bands in 2 other genes in both the conditions manualy treated as well as kit treated DNA..I am using jump start polymerase (hot start polymerase) from sigma, 0.3 uM primers, 2.5 mM mgcl2, 200uM dNTP mix and 50 ng Bs treated DNA when BS treatment done with kit and 300 ng DNA when done manualy. Can somebody tell me what can I do for avoiding those primer dimers?
Thanks
Select better primers.
If for some reason you must use the current set of primers, better optimize your amplification conditions.
Hello
I agree with Tausif Alam , and these links will help you
Good Luck
http://epigenie.com/hints-for-optimizing-bisulfite-primer-design/
Hello Lalita,
From my experience, primer dimer is common in the gels after MSP, due to varying amount of bisulphite conversions, quality of sample,etc. The PCR requires repeated optimization, unlike other conventional PCR. Keep altering the PCR conditions, 0.3uM primers could be a factor, reduce to 0.1uM or even 0.05uM. I had performed most of my experiments at the mentioned concentrations to reduce primer dimers and it had worked fine. Accordingly, the concentration of DNA based on its initial concentration also needs to be altered. Hope this is helpful.
All the best
Dear Lalita,
I recommend you to check primers with software like MethPrimer (http://www.urogene.org/methprimer/) to make sure that your primers is designed correctly. If you use primers for methylated regions only I would suggest to design primers for unmethylated regions too and use them as a control. Also, you can try to optimize temperature for primer hybridization by making different points for annealing step.
Touch-down PCR may sometimes help, but not always. I always do touch down PCR for MSP and CoBRA PCR for methylation analyses. I start with a higher temperature such as if your Annealing temp. is 58, I start from 62 (for 2 cycles), and then come down to 60 and 58 for two cycles each. Then I carry out required cycles lets say 30 cycles at 56. You can give it a go, if you still get dimers, you can also think of increasing your annealing temperature by 1 or 2 degree Celsius such as instead of using 56 deg C, use 57 or 58. That has helped me in the past.
Dear
I recommend the following publication which could be helpful: Methylation-specific PCR: A novel PCR assay for methylation status of CpG islands. Proc. Natl. Acad. Sci. USA 93 (1996). Direct link: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC38513/pdf/pnas01522-0532.pdf
Regards
Marius
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