I want to elaborate a protocol for my qPCR experiement but when i read papers i find that the lenght of the denaturation time in some is 2 min in others it's 10 min. The difference is huge for me so is there any consideration to take into account when choosing the duration?
Hi Ines
Well this depends on the length of your product and the polymerase you're using. You'll find that the leaflet your polymerase came with will have a suggestion for extension time which you can adjust according to your product length.
Good luck
Check with the company where you get your polymerase. I assume that you are asking for the denaturation time at the beginning of a qPCR. Some polymerases need heat activation, such as real-time PCR using Taqman probes from Life Tech needs 10 min for denaturation and Ampli-taq gold from Life Tech needs 9 min. Most Taq polymerases only need 1-2 min. Hope this helps.
Hi Ines,
The parameters for the denaturation phase should ideally be adjusted for the G-C content of the DNA. Since G-C pairs have an extra hydrogen bond, it takes more energy to denature them.
New England Biolabs has a nice online tool to determine the ideal temperature:
http://tmcalculator.neb.com/#!/
There are likely other online tools to help with optimization that you can find with a few online searches.
This paper also elaborates on this principle.
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2727727/
Hope this helps!
As the others have noted, times of 2 mins or more (for denaturation) apply only to the initial "hot start" phase. Most commercial polymerases are supplied with an inhibitor bound to the active site, and the initial step of "heating for X mins" is purely to dissociate this inhibitor so your reaction can begin.
Subsequent to this initial (single) step, ALL subsequent denaturations (i.e. the 95 step in each cycle) should only need to be about 15 seconds, unless your PCR product is enormous...in which case you shouldn't really be using it for qPCR (qPCR works best with short 100-200bp products).
So in short, check the manual for your chosen polymerase (or mastermix if you buy it that way), and use that time.
Thus you should end up with something like this:
Step 1: heat to 95 for 2-10 mins (depending on instructions)
Step 2: 45 cycles of 95 (15 sec), 60 (15 sec), 72 (15 sec)
Step 3: melt curve (if using SYBR green).
- Badges
- Science method