That initial longer melt time allows the entire genome-worth of DNA to open, but later cycles only need to melt a short segment (say 600 bases if that is the segment you are after). That takes only a few seconds.  Another issue: if your program takes longer than you want to wait, try using short extension times.  After all, if your product is only 600 bases, that takes only seconds for the taq to copy, so why do a long extension?  Try some new times, and maybe you will be happy with the results.  Good luck.

My best guess is that it's due to the programming cycle: denaturing, annealing, synthesis. Directly after the initial denaturation stepp no additional denaturation is necessary at all.

Denaturation Temperature and Duration

In question you said 15s and then 30s?

Yes it is due to programming cycles, 1 cycle you can skip denaturation step but its easier to programm all cycles at once.

15sec of denaturation is usually enough.

from neb.com:

• Initial denaturation at 95°C for 2 minutes is recommended prior to PCR cycling to fully denature the DNA
• Avoid longer or higher temperature incubations (unless required due to high-GC content of template)
• Typically, a 15-30 second denaturation at 95°C should be utilized during thermocycling

I do not agree. The protocols do not use longest/most aggressive denaturation times, they use time which is enough to obtain single stranded DNA. For example taq poly is really stable in high temperatures for long time, although protocol says that denaturation time should be 15-30s (of course you can use longer times due to complicated long GC rich DNA). Why you would need longer times when 15-30 is completely enough?

In fact, Taq polymerase is not as stable as you can think. The half life at 97.5ºC is only 5 minutes (!), and 40 min at 95ºC. So the suggested times are the maximum possible to have complete denaturation but the minimum for not killing too much polymerase. In fact, polymerase is so labile after 95ºC that if you add more polymerase to you PCR reaction you will notice that your product increases without adding anything else. That's why many companies have created a hot-start or other polymerases that survive better to long PCR protocols.  

Because I love to run PCR does not mean I love to leave after 19h ! So the shorter the better !! < Sometimes it is less about science than it looks. 

Who did not adjust a basic protocol at least once to have enough time for a real lunchbreak throw the first stone. 

Hi Parvati,

There is a quite a lot of variation in denaturation times found in PCR protocols. Some of it has been derived empirically and others are just a "ball park" figure. But essentially Artur and Edson are correct in that too long at 95oC will cause deterioration of the Taq Polymerase. For a simple PCR with a template of circa 50% GC content I have found just a 10 second denaturation time is quite sufficient.

p.s I love Malick's answer he knows how to plan his day :)

Remembering my good old lab days... I used to take Pfu-polymerase because of the errors Taq made (that was years ago).  I avoided Taq because it was known to make many mistakes. Some people in my lab used a mixture of Taq+Vent  (speed + error-reading).

With Pfu i got a little bit less of an outcome, but the products were always correct (over several years)

Did you compare error-rates of the polymerases available in your lab (or one could order)

PS: I fully go along with Malick now  :)

Edson, Artur,

I agree but still 15 sec is not extreme, I wanted to mention only that.

About Pfu, I had many troubles of getting products or if, the amount was very little so for clonning or mutating I am using simply Taq and then screen for mistakes. Its faster and easier then doing 10x with Pfu ;)

Maybe i didn t explain it right: it was a mixture of Taq and Pfu. The explanation was that Pfu would repair the mistakes the fast Taq makes.

Anyway, if you have good results with a strategy: Never change a winning team...   ;-)

Yes I understood, I tried it as well but the best results (well maybe not the best at the first round) I have with only Taq. I was trying also Phusion and Phusion with Taq.

For me its easier and faster to seqenece more samples then repeat many times PCRs or cloning (but maybe because we have great sequencing facility ;)

and taq is extremely cheap comparing to others ;)

Well, my last thoughts about this issue today:

You didn t mention which buffer you used for the combination: buffer 1, buffer 2 or a mixture (I dont remember what i used, it s too long time ago). This could make a huge difference.

Second: having small yields in one lane of agarose gel is not disturbing. We used to use the Qiagen precipitation colums for DNA. It s no problem to run several dissolved agarose samples through the same column in order to have a higher yield of what comes out when you elute the column. 

Yes, also the prize matters. That s also a good argument, especially when sequencing is inhouse  :-)

Hi,

Actually 15s normally enough for DNA to get single stranded in PCR. 

That initial longer melt time allows the entire genome-worth of DNA to open, but later cycles only need to melt a short segment (say 600 bases if that is the segment you are after). That takes only a few seconds.  Another issue: if your program takes longer than you want to wait, try using short extension times.  After all, if your product is only 600 bases, that takes only seconds for the taq to copy, so why do a long extension?  Try some new times, and maybe you will be happy with the results.  Good luck.

Part of the time required for denaturation relates to the reaction volume. If you are running a 50 ul volume it will take slightly more time to reach 95 than if you are using 25 ul, which in turn will take more time than 10 ul.  And the type and thickness of the vessel wall has an impact.

As others have noted you could keep it at 15s for the first cycle to denature the genome of your target and then try dropping to 10 or even 5s for cycles that follow. Your results will let you know if it works.

I too agree with Jos Schall's logic.