How to get rid of PCR primer dimer - (Mar/17/2003 )

Hi Boutella,
You can try setting PCR at different annealing temperatures ranging from 60 C to 72 deg. Tm looks okay to me. If it doesn't work, titrate the concentration of Mg2+ from 1.5 to 2.5 mM.
Hope it works out for you.

Hi vgupta,
Have already tried the annealing temp thing (did two gradients from 50-70). Thanks though.

Two gradients, does that mean you tried only at 50 and 70? That is not enough. Try at atleast 4-5 deg difference e.g. 60, 65, 68, 70, 72. If it doesn't work, you could try to use formamide to lower the annealing temperature. You could use vent polymerase, it is a good enzyme. What is the size that you are expecting?

No I did a gradient from 50 to 70 (50, 53, 55, 58, 60....) I have tried using Vent (which I normally love) but no luck. I keep getting huge bands at 50 bp. The piece I'm expecting is 1.5 kb. What concentration of formamide?
-bouttela

Bouttela,
50 bp bands are your primer dimers. For that I did touch down PCR starting from 5 deg above Tm and going down to 2 deg below Tm.
So your cycling conditions would be like 95 2 min, (95 1 min, 73 1min, 68 2 min, 2 cycles), (95 1 min, 70 1 min, 68 2 min, 2 cycles), (95 1 min, 68 1 min, 68 2 min, 2 cycles) (95 1 min, 65 1 min, 68 2 min, 25 cycles), 68 10 min.

Sometimes they just don't go away. You may have to design new primers from a different annealing site. I used 2.5% formamide. did you try titrating Mg2+ from 1.5 to 2.5.

I think I might be having the same problem:

My primers Tm are 71.5 and 69.7 oC resp.
I get some small product amplification ~50 bp.
My amplicon should be 220 bp, which is 70-80% GC rich..

So you are saying that I should just try a touch down, starting at 75 down to 68 oC? Without adding formamide or DMSO or Tween or (NH4)2SO4?

The funny thing is that I got this PCR from Zivelin et al:
Improved method for genotyping apolipoprotein E polymorphisms by a PCR-based assay simultaniously utilizing two distinct restriction sites (Clinical Chemistry).

but IT IS NOT WRRRRKN! My PCR reagents and PCR template are working fine though (tested that).

I think you try PCR with and without formamide and titrate Mg2+ which is the most important thing.

Niggle,
I think I figured out my problem by using a polymerase specifically designed for GC rich templates ( Thermalace from Invitrogen, but there are others out there too). I tried the formamide thing but it didn't work.
Good luck and Thanks for the help
-bouttela

I am glad that your problem has been solved. Formamide thing worked for me when used primers with a Tm of 80 deg.