So, I have some primer pairs from a publication that work in silico, but do not give me any PCR product. I tried all annealing temperatures, upped Mg++, did trouchdown PCR-no result. Anyone has any idea what else might be a problem? Some of the pairs have 3-5 degrees difference in Tm. Any feedback is appreciated !
I think Charles is probably right but another possibility is that if your dna is only a similar species or just has a polymorphism under the 3' end if one primer then that primer pair will not work
If the primary pair is published, they must work. Please give some more information about the publication.
Dear Leonid
You qussion is not clear.
Could you send me your primers sequnce??
You could try to do a dna blast to check the sequence.
https://blast.ncbi.nlm.nih.gov/Blast.cgi?PROGRAM=blastn&PAGE_TYPE=BlastSearch&LINK_LOC=blasthome
Copy and past seq of each primer to see if your gene of interest comes up. I have seen published primers be off before. Something happens between the pcr and publication.
Hope the amplification of your dna goes well.
Best,
Charles
I think Charles is probably right but another possibility is that if your dna is only a similar species or just has a polymorphism under the 3' end if one primer then that primer pair will not work
OK, some clarification: so, the primers-all 4-are from a publication and for the 16S rRNA of 4 different species of bifidobacteria. We originally purchased 4 countrol species: longum, breve, animalis, bifidum and I purchased these primers as well because, if and when I isolate new strains, I should be able to confirm them as one of the four species. But first I wanted to use the primers for the detection of the existing controls and none of them work.
Paul, I don't think there may be a mutation in the 16RNA?
And yes, these primers are highly specific in-silico and detect only those species they are supposed to detect.
Most of the Tm-s are around 55C. I ran them above and below and used touch-down protocol as well where I started 10 degrees above the Tm and went below, but no result.
Does it make sense to run annealing higher than 65C?
Thanks for all your feedback!
P.S. Abhijeet Singh there are many primers published that never work ! Publication, unfortunately, does not guarantee their quality. I learned this the hard way, but since I checked these primers in-silico, I thought they might work. I think the best way is to design your own primers.
are you getting absolutely no product...not even primer dimer or the wrong band?
May be worth checking your taq polymerase or taq mixture to make sure the pcr can work.Borrow reagents from another researcher if necessary
I have excluded DNA template and Polymerase: DNAs are good on nanodrop and same polymerse works with other primer pairs. So, it's a bit of a mystery.
It just may have been a copy past error for that pub. Blast the primer sets. If it is wrong order the correct pair.
If it is correct, then it must be something else.
Hi Leonid
As good advises from all,
have you tried for insilico PCR?
Before going PCR you can try insilico PCR by using Fastpcr tool or any other.
Tulsani Nilam : if you really read the answers you would notice the mention of in-silico PCR.
For me (best on experience), the next option is to purchase new 16S rRNA universal primers. Mostly the universal primers for identification work well.
All the best...
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