Hello researchers
the annealing temperature gradient is the routine step in PCR optimization , but why we do not use annealing time for optimization?
regards .
Both parameters are important. An annealing time of approx. 40 seconds up to 1 minute is usually sufficient to allow for oligonucleotide diffusion and templeate binding. Increasing the time usually does not have much effect because oligonucleotides were able to bind and saturate the template. By contrast annealing temperature follows an optimum curve. If this temperature is too high or too low it has a strong negative effect on PCR efficiency. But there are instances when modifying the annealing time can have positive effects. Please see the paper in the attachment.
Cheers, Christian
Article Gradient decrement of annealing time can improve PCR with fl...
Both parameters are important. An annealing time of approx. 40 seconds up to 1 minute is usually sufficient to allow for oligonucleotide diffusion and templeate binding. Increasing the time usually does not have much effect because oligonucleotides were able to bind and saturate the template. By contrast annealing temperature follows an optimum curve. If this temperature is too high or too low it has a strong negative effect on PCR efficiency. But there are instances when modifying the annealing time can have positive effects. Please see the paper in the attachment.
Cheers, Christian
Article Gradient decrement of annealing time can improve PCR with fl...
I agree with the comments given by Christian. Both annealing time and temperature are important.
The annealing temperature is important for oligonucleotide and template binding rather than the annealing time.
Annealing temperature is more important than annealing time in having PCR efficiency
I would keep annealing time fixed (ca. 1 minute) and a play-around with annealing temperature can add a lot to have a optimalised PCR assay.
I agree with Dr. Farsang. The annealing temperature is more important to play around. Using in-silico oligonucleotide analysis, make sure that no secondary structures or primer dimers are formed, which will screw you PCR at lower annealing temperatures.
Goodluck
I agree with all prior answers
- As long as you have sufficient time to make your product: That is 1kb per minute for standard Taq; 1kb per 2 minutes for proof readers more than 5 years old; and quite literally seconds for new the newer generation of high processive polymerases like phusion then the factor - all else being optimised - that will determine success of your PCR is the annealing temp; Thus, play around with this. In particular:
- Try varying the annealing temp from Tm-3C to TM+2C in 1C intervals: In other words 6 separate PCR reactions where the annealing temp varies in 1C intervals, TM-3C, TM-2C etc. Generally, You will find that Tm-2C is optimal in terms of yielding a strong specific (single) band. Above that temp, i.e. @ the TM and above you will obtain a specific band (in the case of new generation polymerases; traditional Taq often doesn't work @ the Tm) but it is invariably weak. Below Tm-2C you will also obtain a strong band but run the risk of non specific bands as well
- If not sure, to determine primer Tm see document attached. Also pay attention to the last 5bp of your primers and preferably terminate with a double purine; that is GG, GC, CC, CG to maximise PCR efficiency and thus your chances of obtaining a strong band
- Very occasionally, Primers fail to yield a band at all: In the absence of double purine termination and other factors enunciated in the attached doc dropping the annealing temp down to Tm-5C can sometimes make the difference between a single specific band and no band at all
- Furthermore, for such primers/Reactions, adding an extra 0.5mM MgCl(2) to the PCR reaction, e.g. 2.0mM to 2.5mM (final concentration) can make the difference between no band at all and a single specific band. Alternatively, I have found touchdown PCR to be most effective in these scenarios. See attached: In this case perform touch down between Tm-5C to Tm+2C in 1C intervals
See by way of example;
95oC 3 min x195oC 15 sec
63oC*1 30 sec ( - 0.5oC/cycle) x14
72oC 3 min
94oC 15 sec
57oC*2 30 sec x25
72oC 3 min
72oC 10 min x1
4oC ∞
*1: Initial Ta = Tm + 3oC
*2: Final Ta = Tm – 3oC
Because Annealing temperature is the critical factor that normally determines the binding of the primers with the DNA/cDNA. Few seconds are enough for the two to anneal and begin the amplification.
Although, some do optimize annealing time !. But, base on my experience it has no much effect on the PCR amplification success.
In my optimization works so far, I find time and temperature indispensable. That is the reason the use of optimization is vital in order to know the optimum conditions sine neither time nor temperature can be infinite.
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