I'm designing specific primers to clone a small (~298 bp) fragment from a plasmid and introduce restriction sites. Both forward and reverse complement primers are 27 bp, no hairpins, non-self annealing, etc. The complimentary 18mer sequences which will initially anneal to my template have a Tm (nearest neighbor method) of 56°C (forward) and 56.5°C (reverse). However, the Tm of the entire primer including restriction site and extra 5' bases is higher at ~67°C for both. Many sources say to just use the initial lower Tm to determine a single annealing temperature throughout the run, while other sources suggest increasing the annealing temp after 5-10 rounds to account for the introduced bases with higher Tm. Has anyone had any experience with optimizing along these lines and if so what would you suggest?
I would agree with Jacob. During the first couple of cycles in PCR, the majority of amplification will come from the original template. Since the sequence for the restriction site is not in the original template, then the lower annealing temperature would be applicable. As you generate PCR products, the products will include the sequence of the restriction site and the higher primer Tm value becomes more applicable. Using a higher annealing temperature after a few cycles may not generate as much product. But the products you generate will be cleaner.
Increasing the annealing temperature tends to give cleaner PCR products, at least in my hands. Hav done so for many years.
I would agree with Jacob. During the first couple of cycles in PCR, the majority of amplification will come from the original template. Since the sequence for the restriction site is not in the original template, then the lower annealing temperature would be applicable. As you generate PCR products, the products will include the sequence of the restriction site and the higher primer Tm value becomes more applicable. Using a higher annealing temperature after a few cycles may not generate as much product. But the products you generate will be cleaner.
Dear Michael,
Initially you can try the low annealing temperature and if you observe non-specific amplification, you can increase the annealing temperature after cycle 10.
Best
Thanks guys, I'll try increasing the temps after a few cycles as you all suggest.
I introduce restriction site and amplify the PCR product ( up to 6 kb) for cloning frequently. I only consider the annealing temp. of primer that directly bind with template DNA and never had any problem. In case, if I get non specific product for longer fragment, I usually go for 60 and then extension at 68 with increase few sec every cycle . If the problem still persists, I usually excise my desired band from the gel.
For what it's worth, I routinely generate PCR products with additional terminal restriction sites in the primers (20 bp specific for the target plus 10 bp including the restriction site and additional bases to ensure proper digestion of the PCR product). I've never worried about changing the annealing temperature during the PCR run, just stuck with the original set temperature (58C as a rule), and never encountered any problems. So, it may not be necessary to complicate your protocol.
I've tried both strategies and in all cases I got the same good result. Nevertheless, to ensure the maximum number of copies of your template DNA with the restriction sites added to the ends, I would perform 5-10 cycles with an annealing temperature of 52-53ºC and then, 25-20 cycles with an annealing temperature of 63-64ºC. In the first cycles you amplify your template DNA together with some incorrect or incomplete copies. When you increase the annealing temperature, you are ensuring the amplification of those templates containing a perfect copy of your restriction sites. I think that this is always recommended, unless you have very long tails (30-50 bp), in which case it is probably very difficult to be accurate calculating a correct annealing temperature.
I do not think it is necessary. I did hundreds of clonings, never had any problems sticking with a certain temperature. I start with my standard 52C without taking into account the annealing Tm of the primer.
To generate recombineering templates, you generally add 40 to 50 bp recombineering homology to the PCR homology, totaling ~70 bp. Even in that case, I never increase the temperature. I did hundreds of those too.
Sometimes, a reaction does not work, then playing with the annealing temp over a small gradient solves the problem.
However, I do like the idea of increasing the temp much. It is the first time I hear of this. I will certainly keep it in mind for problematic situations. After having done so many clonings, problems still arrive once in a while (you never become the full 100% proof expert), so any tip to reduce the frequency of these is much welcome.
I can't say I've tried increasing the annealing temperature after the initial cycles, but here we routinely do many PCR tests with restriction sites coupled to the primers and have always just used the lower annealing temperature. It's always worked for us, which is probably why nobody has ever tested the other protocol. Like the others have said, increasing the annealing temp will probably just increase your specificity, but if your built your primers to avoid non specificity, then you should be good no matter which protocol you follow.
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