Hi, I have been trying to decide on the best annealing temperature for the F/R primers using a sample. From left is the ladder, 61c, 63c, 65c, no template and the ladder. The expected product size was above 150bp and the shortest band of the ladder is 100bp. What could have possibly gone wrong? I am pretty sure I didn’t add anything to the no template control!
Dear Noyan Nemati
the bands that you see in your gels are associated to the primers and for these reason are smaller than 100bp.
the bands that you do not see any other band is not clear to me. To give you some suggestion we need to have more information about your gene sequence, primer sequence, PCR cicle and PCR reaction assembly, but from your gel it seems that something is missed in the reaction or the primers are not annealing in the template, since your PCR are all negatives.
best
Manuele
Dear Manuele Martinelli
The only thing I did and feel was wrong was that I added ~ 5ml of buffer to the top chamber after loading the ladders and sample. This caused the content of the wells to move out a little bit.
The primers are ~23bp with 6.82ng/ul each in total of 16ul. Though when you say the bands could be primers would they be this intense at that concentration?
thanks
this looks like primer dimer where one primer finds a homologous region in the other primer, anneals and then extends.The product is short so melts easily and amplifies well so takes over a pcr reaction until all the primer is used up . There is often no proper product when you have primer dimer. Try using a hot start polymerase or redesigning the primers
Dear Paul Rutland
We are using MyFi DNA polymerase (hot start). And around 2 years ago these F/R were used in the lab - kept since then. Both primers though include CC,AA,TT,GG in them. Since I can't share the primer sequences, are there any tools that let me investigate the possibility of dimerization?
Thanks
try the oligo analyser tool at idtdna
OligoAnalyzer Tool - Primer analysis and Tm Calculator | IDT (idtdna.com)
https://eu.idtdna.com/pages/tools/oligoanalyzer?returnurl=%2Fcalc%2Fanalyzer
You could also try using less primer (half or a quarter) your current amount but it may be that the primers are degrading if they are dissolved in water not TE in which case a new primer synthesis may be needed. You could try both using less primer and raising the pcr mix (without enzyme) to 80C then adding the enzyme as a manual hot start then start cycling on one or two samples to see if a hot start can work just to check that the polymerase has not lost its protection and has become partially active even at low temperature before enzyme heat activation
Dear Paul Rutland
I just realized I have access to IDT analyzer and had ago - there are 13 possible ways that heterodimer can be formed, and their Delta G are from -4.4 to -1.3 kcal/mol. Some have up to 8 'complementary bases for dimer structure'. I think heterodimer formation is likely.
IDT reckon anything with deltaG higher than -9 is a possible primer but 8 complementary bases sounds like a problem even if -4.4 should work so possibly running a dmso gradient from 1% to 9% to minimise secondary structure could be worth trying if primer redesign is a problem. Of course also check the od260/280 ratio of the dna that you are amplifying
Dear Paul Rutland
When the primers have high complementarity does longer centrifugation of master mix before adding template make them forming the bonds.And thank you kindly for your time and advice.
The formation of PD depends on the presence of active enzyme together wuth anneaked primer so if the hot start enzyne js good then the first step of degradation at 94c will split the primers into single primers again so unless you are spinning for a very long time and the samples are heating up I would not expect centrifugation to make any difference. With cold start enzymes the longer and warmer the master mix remains before starting pcr the more likely it is that PD forms
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