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Primer Annealing Temperatures - (Aug/21/2012 )

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I have two different annealing temperatures for my primers (59 and 55). At what annealing temperature should i set when in the PCR when using this primer set ?

Thanks for the help


I use this site to calculate the Tm :
There are many algorithms around leading to different results. With the Tms determined here, I take the average - 5 oC for the annealing when I am using Taq polymerase (30 sec anealling step)
Take care that if you have overhangs (additional sequences you want to add to your sequence through PCR) you need to calculate Tm only for the matching part of the primers. For this purpose I use Phusion: higher Tm of both primers (matching sequence) + 5°C; touch down -0.7°C / cycle; 15 sec annealing step; this for 14 steps followed by another 6 steps for which you have: lower Tm of both primers (calculate Tm for full sequence) (15 sec per step)



Too complex for me. I anneal at 55C.


Start with 55, if the product in unspecific increase annealing temperature by 2 degrees at a time. that should work :)

-Ameya P-

Step 1: anneal at 55
Step 2: (optional) do a gradient PCR annealing from 50-68
Step 3: Throw primers away, redesign primers.

Life is too short to deal with marginal primers.


I usually do..
Step 1: anneal at 60
Step 2: 5% DMSO
Step 3: forgot.. long time ago


why redesign primers? those are perfectly good primers. I did a PCR once with primers one at 46 and the other one at 71 (long story, but I couldn't design them better no matter what I did, and I struggled quite a lot). Worked perfectly....but I'm the one with a too complicated PCR program Designing primers with the same annealing temperature is so old-fashioned. What the PCR cyclers can do nowadays for you together with what we know about PCR can easily solve the problem of the PCR with different annealing temperatures. And btw: 4 degrees is nothing.

I am also all for the gradient annealing suggested by phage434. However, not all the labs invest in such a function of the PCR machine...(my current lab surely didn't invest in this option no matter how many times I cried for it ) But if your PCR machine can do gradient or touch-down: learn how to use them, they will save your life.



Could you share the program you then used to get primers to work? I never violated the recomended 2 deg difference, even when I had some pretty awfull sequences too (but I work on nonextreme GC% templates), so seeing different approach would be interesting. Thanks.


I will post here the entire Phusion protocol I am using from mastermix to agarose gel:
1. Setup on ice in a 0.2 mL tube the mastermix for PCR according to the table
below. Thaw all non-enzyme components at RT, mix by short vortex and collect
by short centrifugation.
(75 μL; split into 6x 12.5 μL)
-plasmid template
(sequencing or column grade
plasmid preparation)
x μL (5 ng / 1 kb plasmid template)
-MilliQ H2O
(PCR quality = autoclaved in a bottle that was rinsed several times with MilliQ because you never know who made their Mn/Mg salt solution in that bottle before you; once I have a bottle I can trust, I always reuse the same bottle for preparing PCR water all the time)
47.75 μL - x μL of plasmid template
-5x HF buffer 15 μL
(final 1x)
-X_fwd primer
(5 μM)
6 μL
(final 400 nM)
-X_rev primer
(5 μM)
6 μL
(final 400 nM)
(10 mM each)
1.5 μL
(final 0.2 mM each nucleotide)
(2 U/μL)
0.75 μL
(final 0.02 U/μL)

2. Split the mastermix into 6x 12.5 μL and run all samples in a PCR cycler using
a 10°C temperature gradient (use columns: 1, 4, 6, 7, 9, 12; use the cycler
option menu to calculate the corresponding temperatures). (this is for the Eppendorf PCR machine; the one for 96 samples with gradient included; check for your own machine how it works)

PCR program ('' means sec and ' means min):
(20 cycles; 105°C (works also with 99°C) heated lid and set to PAUSE)
ID – initial denaturation 30’’ 98°C
D – denaturation 5’’ 98°C
A – annealing 15’’ = higher Tm of both primers + 5°C;
gradient +/-5°C;
touch down -0.7°C / cycle
(calculate Tm for matching sequence)
E – extension 15’’ /1 kb 72°C
go back to step 2 and repeat 13 times
D – denaturation 5’’ 98°C
A – annealing 15’’ = lower Tm of both primers
(calculate Tm for full sequence)
E – extension 15’’ / 1 kb 72°C
FE – final extension 3’ 72°C go back to step 5 and repeat 5 times
S – storage 8°C
3. Load all samples on a 0.8 % SB agarose-gel (7.5 V/cm; 40 min) (for me SB buffer for running agaroses is the ideal buffer; TAE is an ancient buffer developed because they did not know better than Tris at those times. But this is a completely different story :). Check for optimal annealing temperature among your 6 samples and repeat PCR with that temperature if more product is needed.

BTW: when repeating PCR never use more than 20 uL per 200 uL tube.

This is called Phusion-never fail PCR and was developed by my supervisor during master thesis, Dr. Alexander Schenk. Enjoy you PCRs :)



Little note: since in my current lab I do not have a gradient PCR machine, I skip the gradient and it still works; just use the middle temperatures as described in the protocol. Still my PCR never failed:)

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