PCR anealing temperature question - (May/07/2006 )
One primer has Tm=93, another has Tm=101, The length of both primers are 30 base pair. The product I want to amplify is about 2.2kb. I have tried anealing temerature at 58, 65 and 68. I got a very very week band. Can anyone give me some suggestions? Thanks a lot
Yue
your tms are higher than your denaturing temperature. you need to find a way to reduce the tms of your primers.
I think it would be good to design new primers, but you could also use a polymerase like Phusion if you can only get the temp down a little...you can melt your DNA at 98 and get good results. but the one that has a Tm of 101 still would have to be lowered
Or maybe an additive like DMSO (used at 5% v/v, I think) or betaine (the monohydrate, not the HCl; used at 1M final concentration, I think) would allow the reaction to work.
But, I agree that your Tm's are way too high -- must your primers have such high Tm's? Can you make them shorter?
I don't know how you setup your PCR reaction but to the best of my knowledge NH4+ ions reduce annealing temperature so you can try using PCR buffer with ammonium sulphate. Write to Fermentas, they have such buffer and they may offer some advice, if they do and it works out, please post your results so that others know if this has worked.
They suggested me same a while ago but I didn't try yet.
I doubt that's your actual tm for a thirty base primer. What formula are you using?
I would try 48-55 for your annealing. If that doesn't work, try adding 5% DMSO or 500 mM Betaine.
-Matt
Thank you for all your comments. The Tms were calculated by Invitrogene when I ordered. I added DMSO and got a slightly stronger band. I think I didn't design the primers very well. Anyway, I will use what I got to do cloning. Thank you.
Yue
Yue
Hi. Unless your primers are poly G or polyC, I can't see how you'd have such high Tms. Work it out for yourself, and I think you'll find a much nicer number (can't always trust what you read, eh?). 2C for an AT pair, 4C for a GC pair. An alternate to DMSO is betaine.
There might also be an issue with the difference between the Tms. 8C is probably too much difference: go for something ~4C and you should have no problems. (I had to do PCR on DNA that was 85% GC content, so trust me, it'll work).
That formula is not correct.
Use either:
Tm = 64.9°C + 41°C x (number of G's and C's in the primer - 16.4)/N
OR
Tm = 81.5°C + 16.6°C x (log10[Na+] + [K+]) + 0.41°C x (%GC) - 675/N (taking into account salt concentration)
http://www.promega.com/biomath/calc11.htm
Where N = number of nucleotides in primer.
If you simply provided the G/C content by percentage or the primer sequence, I'd have a good idea.
Good luck,
Matt
dear Yue
can u plz calculate the Tm according to the following equation plz
60.8 + 0.41 x (GC%) - (500/n)
n= no of base pair of the primer