No bands despite different primers and conditions and TAqs - (Oct/21/2013 )
I am trying to get a 150-200 bp PCR product from genomic DNA (for pyrosequencing). However, after trying different annealing temperatures (52-62 C), extension times (30-60 s), MgCl concentrations (2-3), primer concentrations and 2 different TAQs, I still don't get any visible PCR products on a 1.6% agarose gel. I have a positive control (1200 bp), which always works perfectly. With 2 primers flanking my region of interest, I got a PCR product which I sequenced and which seems to be correct.
Has anyone ever experienced something like that and any further advice what I could do about this?
Thank you so much!!
If the region has a high GC content, or is likely to form significant secondary structure, then it may be necessary to add a secondary structure inhibitor such as DMSO or betaine.
As you are getting nothing, it is hard to trouble-shoot, so I would suggest that you try some lower annealing temps (as low as 40) and if you are using normal Taq, definitely try using 1.5 mM MgCl2, as it works well at that concentration. Higher concs of Mg are normally only needed if you have an excess of dNTPs in the reaction.
Many thanks, will give DMSO another try. If I figure anything else out I'll get back here. And if there are any other suggestions - welcome :).
Another thing that can be a problem is contaminants/inhibitors in the DNA that are causing the reaction to fail (might only fail for some particular reactions) - try diluting the DNA 1:10, 1:100 and 1:1000 and see how that goes.
Sometimes primers simply don't work. I'm now of the opinion that if primers fail more than 2-3 times after trying gradient PCR and DNA dilution, I choose diferent primers. Life is too short to fool around with poor primers, and I've found that a second set almost always works.
Many thanks for your advice! FYI: Finally, the 5th forward primer gives me a robust result but only with the 2nd TAQ, after adding DMSO + doubling the primer concentration + 3-fold MgCl concentration and with 50 cycles @ 62C.
Took a while to figure this out...
Be aware that 50 cycles is an awful lot - at this point you could easily be amplifying contaminants.
I strongly agree with Bob1. Anything beyond about 35 cycles is suspect, unless you are working in extreme conditions, such as single cell PCR.
New, different, primers! Or perhaps what you are trying to amplify is simply not there. I don't know what your template is, but if it has extremely low GC sections of 30 bp or so, it is sometime necessary to lower the extension (not the annealing) temperature to 63-65 instead of 72.
Thanks for your advise - since I am pyroseqencing my product and it works nicely I am not really worried about amplifying contaminants ;). I often increase the number of cycles to increase the yield - if the reaction is specific I haven't had any problems with that so far.