Good afternoon all!!!
Please what might be the cause(s) of inability of different primers designed for a specific product length to give such output after PCR and gel electrophoresis?
The different primer pairs keep having a product length less than 200bp as against 790bp.
Thank you
your genome or amplimer probably has a similar sequence to one or both primers.Maybe a pseudogene or other gene family member. The smaller product will melt easier and will almost always dominate the pcr reaction. It may be that your pcr specificity is too low allowing the other amplimer to form. Try increasing the annealing temperature ideally with a gradient pcr or possibly adding 25up to 8% dmso to the reaction mix may solve this problem
To begin the troubleshooting you should run a primer blast with organism-specific parameter (https://www.ncbi.nlm.nih.gov/tools/primer-blast/). This will give you the potential targets for your designed primer sets. Further, if there is any target that is below 200bp, it means your primer is binding non specifically and for that matter, you can follow the excellent suggestion provided by Paul Rutland
Another reason for this result can be intermolecular base-pairing between your primers. You may check the 5' and 3' homology of your primers.
All the best
Umer Saleem Bhat Thank you so much
Paul Rutland Please could you explain "adding 25up to 8% dmso to the reaction mix may solve this problem"
I am lost
Sorry Semiu Folaniyi Bello and Umer Saleem Bhat the 5 and the % are on the same key on my keyboard. I meant to run samples containing from 2% to 8% dmso . Increasing DMSO increases the specificity of priming and reduces secondary structure of the amplimer and target dna so if the short amplimer is non specific there may be an amount of dmso that stops it working non specifically while letting the specific primer anneal and amplify. Above about 10% dmso the polymerase starts to be destroyed but generally dmso is good at cleaning up amplifications. It works rather like increasing the annealing temperature in pcr.
Thank you @Paul Rutland. Please would the dmso be added to the PCR mix?
Thank you once again for your time
For the first dmso gradient it would be easier to make up the pcr mix with the dmso replacing some of the water in the mix in each tube but if it works then the amount that works best can be included in the master mix Semiu Folaniyi Bello
- Badges
- Science method