I am trying to amplify UTR region of a gene by desiging primers against genomic sequence. Melting temperature for all primer sets is 59. I tried with different master-mixes (Robust, emerald, Fast, AmpGold) but not getting amplification. Only one set of primers gave result while others are not giving any bands. I tried touch up, touch down and changed the annealing temperature but still not getting any band. Please suggest me any option.
I will list some of the obvious controls/ideas which you probably performed or though about already:
- are you sure gene is present in the material you are using? (I assume you have a genomic extract and a species with well-known genomic sequence, is that correct?)
- what is the position of the primers that work compared to others? Maybe there is a deletion in your sequence?
- did you check primers compatibility with a tool like primer3? http://bioinfo.ut.ee/primer3/
- do you get a product with any other control primers with similar Ct?
HI Katarzyna,
I am using human sample and it contains that genomic sequence. Primers are overlapping to cover whole sequence. I used blast to check the compatibility of primers and designed it with blast only. I tried with different samples but not getting bands.
Blast is not sufficient, that will only check if the primers are specific across the genome. What you want to check is that they don't have any internal structure or pairings that can impair the PCR product formation. Check the link I pasted above, that tool does just that.
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