primer dimer - proplems (Jan/21/2010 )

Hi all:
still the same proplem, I had run PCR for amplifing specific gene, and i got very nice clear band at the expected site, 358bp, then i tried to run more sample at the same concentration, unfortionataly, primer dimer appear, with no product, my protocol as the following:
1- buffer1x=2.5ul.(Fermentase sdn Phd)
2-Mgcl2=2.5uM.(Fermentase sdn Phd)
3- dNTP=0.38uM.(Fermentase sdn Phd)
4-Fp=20pmol.
5-Rp=20pmol.
6- taq pol= 2U, 0.1ul (Fermentase sdn Phd)
7- DNA temp, 200ng.
8- DMSO, 10%,,and dH2O. total volum=25ul.
1st denatur,94c-4min, denatur,94c-1min, anneling,55c-1min, extens72c-1min,last exten,72c-10min. total cycle30.
the only dNTP i changed after i got the product, because it finishes, the new dNTP was the same concentratioan as before,,,,,how to overcome this primer dimer.

maxmix on Jan 21 2010, 06:57 PM said:

Are the dNTPs that you use pre-made by Fermentas at the required combination, or do you get each individual dNTP and mix them yourself??

If you mix them yourself, it seems as though the only thing you changed between the successful and unsuccessful PCR was the dNTPs that you made? I'd therefore suggest that you throw out the dNTPs that you made up that didn't work and make them again/check that they work using a reliable PCR you know always works!

Personally, I use a higher dNTP concentration than you - is 0.38uM final concentration? ... My dNTP mix is typically: each dNTP is at 100mM, which I then combine to create a total dNTP stock of 4mM. When this is diluted in the PCR reaction, the final concentration is 0.2mM - which is 200uM, which is much higher than yours!

Hope this helps!