Hello dear scientists.
Can the specificity of primers change over time or depending on the sample? It looks like the primers I used in my recent PCR to target exon13 of CFTR gene on chromosome 7, actually amplified a different location on chromosome 15. The gel looks like ladder with two sharp bands.
I suppose if most of the specificity of the primer was at its 3'end and there has been nuclease degradation due to primers being dissolved in water and not in TE then you might get specificity elsewhere in which case re ordering primers is needed. Even if this were the case I would expect to see some correct product so it seems more likely that there has been a labelling mistake and that you are somehow using chr15 primers mis labelled as cftr. Primers are cheap best to order fresh
Paul Rutland Thank you for answering. I used some old primers that were mixed with water. They were labeled as "exon13 of CFTR primer" by the company that produced them. Perhaps the initial design is wrong. As you mentioned, I will design and order new primers.
It is possible for primers to amplify different sized products from different templates. That's actually a feature that folks use to genotype for Indel mutations, copy number variants, etc.
But, given the uncertainly in the tube label, I agree with Paul Rutland that ordering new primers is the cheap & easy fix.
Good luck! Let us know if that solves the issue for you.
The company probably put the primer sequence on the vial. It might be worth Blasting the primer sequences just to check if they have any relevance to cftr in spite of their name might clarify the problem
Out of curiosity, how do you know that the other band is from something on chromosome 15? Did you sequence your PCR product?
You can also get different results if you use primers designed for one organism to amplify from a different cultivar or species.
I agree that checking the primer sequences and just ordering a fresh set are great ideas.
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