Possible to use two different annealing temps? - (Jun/13/2007 )
Hi! I know that you should use primers with about the same Tm, but theoretically is that really necessary? Can you not have two annealing temperatures, one for each primer and have the highest first?
An example of what I mean. Primer F have a Tm of 79, and primer R of 65.
And I use the following cycle:
Denaturation 95 1 min
Annealing 74 30 sek
Annealing 60 30 sek
Extension 72 1 min
Is this not possible? If not, why not?
If I got som unspecific bands, that it ok, I can cut the right one out from the gel and purify the product.
An example of what I mean. Primer F have a Tm of 79, and primer R of 65.
And I use the following cycle:
Denaturation 95 1 min
Annealing 74 30 sek
Annealing 60 30 sek
Extension 72 1 min
Is this not possible? If not, why not?
If I got som unspecific bands, that it ok, I can cut the right one out from the gel and purify the product.
Hi Ammie,
yes you can use primers with different Tm but the difference should not be excessive. I'll say not more than 10 degree.
however, My cyles are different from your like this..
1. 94 for 2 min
2. 94 for 30 sec
3. 60 for 30 sec (Ann)
4. 72 for 1 min (extension)
5. go for cycle 2 for 14 times.
6. last 72 for 2 minutes
7. 6 forever
8. end
if you like .. you can download Primer Sets and PCR Manual.. very useful.
http://labclone.blogspot.com/2007/06/prime...pcr-manual.html
cheers,
Amit
Ammie I don't recommend to use 2 anneling temp in the same cycle, you can get some unspecifiy products. Whe design primers they should be no more than 10 degrees of temp difference. But if that is what you have try to use the average of both temp. That will be 72, but that's the extension temp. and must be a difference between the anneling and extension. So you can try at 68C, but sincerily the better is to make another primer. change the one of 79C for another with a lower temp. I'm guessing but I'm almost sure that the one with the higher Tm has a more than 60%GC. If more that 60%GC then that primer must no be use, that's a rule of thumb when designing primers.
Why don't you try different programmes and see what you get? Empirical research, the backbone of science (theorists can help, but they tend to be a strange lot).
The one with the higher Tm is much longer than a "normal" primer (because I want to introduce some nt's in the end). I could of cause design a long matching primer, but I will use this one in many combinations and hoped that I could use it with the primers I have.
So I try som different programs. Just wanted to know if it was hopeless or not.
Unspecific products do not have to be a problem, because I anyway gelpurify my product before I will clone it.
Thanks!
If you look just at the matching part of your "longer than normal" primer, what's your Tm then?
It is around 50. But that does only matter for a few first cycles, doesn't it?