We are having problem to PCR Rice SSRs- RM72, RM163, RM206..., others like RM21, RM136 worked. We use Hot start Taq 2X Master mix from New England Biolabs (#M0496). Tried annealing primers at -5 to -10 degrees below the Tm. No bands at all. Your suggestions will be highly appreciated.
Thanks.
yes. working with hot start taq needs high annealing temperatures. you can try gradient PCR with 60. 63 and 66. This usually works.
All the best!!
In general my impression is that annealing temp. below the what is suggested for the primers is working. We are using NEB hot start Taq and the software they provide also calculates lower annealing temp. Thank you Anupriya for your suggestions!
In case you got product for RM21, RM136 if you have done PCR with these primers earliest to others (which did not amplify), I would like to suggest for quantification of DNA sample. Since DNA isolation from grain sample may not give you sufficient DNA, this problem might be arising. Or else if you have DNA ( not degraded), then try Touch Down PCR, it may work.
Best wishes
That particular polymerase system uses reversible aptamer binding to produce the hotstart activity; this one has an activation temperature of ~45C. If the Tm is this low, then the polymerase may have difficulty extending. However I doubt this is the problem. In my experience you're facing one or both of these issues: carbohydrate contamination from the seed tissue (very hard to remove from seed material, especially if sample size is limited) or simply that the primers are poor quality in their design. I frequently see people fail with certain RM-series SSRs even with good quality DNA. In all cases this seems to be simply that the primers have only marginal functionality, making them very sensitive to minor fluctuations in other parameters such as DNA quality, quantity, Tm etc., some of which can be very hard to control or even identify. The only robust solution is use better markers, or at least better primers for these markers. If you really must persist with these markers/primers, try testing your DNA purity for CHO and RNA contamination (check your A260/A230 is >2.1 and A260/A280 1.9>Absorbance>1.7), quantity is >30ng/uL, and try a touchdown as suggested by Pritesh Roy. You can also go through the usual optimisation of the whole PCR process: systematic variation of Mg, dNTP etc. concentrations, as well as trying other additives like DMSO and sucrose. Hope it works!
I also suspect that culprit is the template DNA, possibly contamination with CHO and RNA as suggested by John and Pritesh. We had checked DNA quality with Nanodrop and it was largely okay. However, we recently noticed DNA degradation over time for some samples, but don't know why. We tried very low annealing temp (46 degrees) as higher temp yielded nothing and it turned out amplification of very large segment (~1000-1200 bp) with nice and crispy bands but no expected SSR alleles. We will try touchdown protocol and report what comes out. Thank you everyone for your suggestions.