I have synthesized a gene specific primer for amplification of my target gene. However, I am getting amplicon of only 100 bp rather than 210 bp (target amplicon size). So far I have tested so many optimization conditions for it but still getting non-specific band. I have cross checked the primer pair for non specific binding in my extracted DNA, it shows no complementary with any other region except for my target region.
Kindly help me out in this
how big is the amplimer if you BLAST the primers?
Are your template samples and primer design templates the same.....what I mean is did you design primers against genomic dna but are now amplifying cDNA which could make the amplicon smaller once the intron has been removed
Also, have you double-checked the sizes of the bands on your ladder? Based on the size of your primer-dimers, I'm guessing that last band on the ladder is more than 100 bp.
Good luck!
Dear Aliya,
It happened to me as well. However the simplest way to tackle this problem is PCR conditions. Try to find out PCR conditions for your gene from any article or use fewer (25 may be) cycles with annealing temperature (45 for 5 sec). Thats what I did with my bacterial samples. Hope it helps. Good luck and keep trying.
Paul Rutland Thank you so much Sir for you informative response.
I have synthesized the primer pair based on the exonic sequence of my target gene. So I was supposed to get amplicon of 210bp.
What else I could do to resolve this issue?
Your kind suggestion would be appreciated Sir!
Shubhra Singh
Thank you so much for your informative response.
I will run PCR as you have suggested.
Katie A S Burnette Thank you so much for your informative response. Yes Mam I have cross-checked its size. It should be of 210 bp.
The pcr does look very clean and strong so following up on Katie A S Burnette suggestion try running one sample and someone elses dna ladder in case the ladder is made up in high salt buffer and is running slowly. Can you attach the primer sequences and species for the gene please?
- Badges
- Science topic
- Similar topics
- Analytical Chemistry
- Extraction