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 the better for PCR optimization

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  

Both are important, but the annealing temperature (too low or too high) can interfere in the specificity of your reaction as well as primers disponilility, specially if you didn't check properly secondary structures (hairpin, dimer, etc). Regarding the annealing time, a rule that I heard a lot of times, but unfortunately I can't say for sure, is that you should have one minute/kb. Of course if you keep too short time for a big fragment, you may not have the most optimal conditions (or great final concentration) in the end of the reaction.

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.

Annealing time can be depend on copy number of gene?

its depend on your product 

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.

HI, EVERYONE,iasked in general whate does the best time forannealing the metal oxide/rgraphene oxide .