Hello Academics
I am in a really weird situation now and I'd really appreciate your help in this.
So I had a ordered a set of primers and did my PCR (using standard (ABM, TAKARA) first and then pfu) and had proper bands and all was well, but now after about 4 months, I need to do the same again, but unfortunately, I get absolutely no bands whatsoever. I thought my primer had degraded (even though I store it in -20 deg freezer) so I re-ordered and tried again but still didn't get any bands. Did standard PCR with ABM polymerase, and also with Takara but still no results. Did a touchdown PCR, still nothing. I even re-made my cDNA template. Am using everything as same as I had used before, so I don't understand why the PCR has suddenly stopped working. Any help or suggestions would be very appreciated. Thank you in advance.
the first set of primers may have degraded if dissolved in water not TE or possibly if stored near the cooling coils of a self defrosting freezer so subject to temperature changes in the thawing cycle. Does any other primer set work on your cDNA?. Check the sequence pf the primers again, Try a pcr with too much primer in case it is mainly degraded but some still exists. Also check the quality of the cdna using housekeeping gene amplification. Check the paperwork or phone the oligo company to check that the synthesis of the oligos was perfect( good yield and trityl measurements were as expected). If the cdna looks good I would order primers again as it is quick and cheap
Paul Rutland
Sir, i checked my cDNA and it is fine and I have also re-ordered the primers and did PCR again but still no outcome. Yes my other primers still work on my cDNA. Am just so confused right now on how to progress. Should I just give up on this primer and order another?
Apart from your primers there can be several other things which can be messed up causing your reaction to not work. Check the quality of the dNTPs and the buffer condition you are using and whether they have been stored properly. Sometimes slight variation in composition can lead to well working PCRs to stop working. Also, check the thermocycler in your lab, which although displays proper temperatures, can sometimes not function properly causing temperature fluctuations. Try your reaction in some other instrument if possible.
Sukanya Chakraborty
Hi. I have checked the dNTPs and all my other reagents. I even used all new packs of everything. Still got nothing. As for the thermocycler, if it were to be problematic, wouldn't all or at least most of the PCRs be messed up? Confused :(
The problem does seem to be between the primer and the sequence. Is it possible that the new cDNAs are from a population that has a SNP under the 3' end of one primer that was not there on the original cDNA ( maybe check dbSNP database). In this case the pcr will fail unless the annealing temperature is dropped so low that it will anneal and amplify. I would try if possible, new primers on the old cDNA and old and new primers on the new cDNA but drop the annealing temperature by 6C, The result may be dirty but can be cleaned up if it works at all. It probably would save time if you ordered new primers while troubleshooting is in progress as they are very cheap.
Are the primers that worked from the same company as the primers you ordered the primers that work. Also, did you do a check on the Tm and delta G for each of the primers in your set? Possibly there was a mistake in dilution of the primers (incorect molarity). Finally, your template, is it possibly contminated with phenol? Do you have a pair of primers that amplify another segment of the cDNA.
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