primer dimer

[mariaiqbal]

hi.i am getting dimers along with my PCR products,,one of my collegue told me to reduce amount of primers as i use 1.25microlitre of each primer so i start using 1 microlitre of primers but still getting dimers,,issue is not in PCR but when i do RFLP as one of my band size is 47bp than i cant distinguish weather it is dimer or my band ...tell me what to do???

[mchlbrmn]

You haven't said what levels of primer you use, but the normal range is 0.2-1.0 uM, with 0.3-0.5 uM commonly recommended. Reducing primers may help, but personally what I would do first to eliminate primer dimers is:
1. Redesign primers changing the exact sequence of the 3' end to avoid 3' ends of the same or for+rev primers annealing to each other. Adding or removing or shifting the primers by 1 or 2 nt can often make the difference.
2. Raise the anneal temperature. (Or, add a cosolvent?).

[mariaiqbal]

actually its not in my hand to redesign primers,,any other possible thing to do???

[mchlbrmn]

#2 above?
Also, like they said, you could reduce the primers, but do it by 1/2 or 1/4. 0.8x isn't much of a change. However, stay within the concentrations I gave.
If you look at the primer sequence, see a homology of 3' ends, you can show it to the appropriate 'hands' and suggest a modification of + or - or shift a nt. or two.
You could also run out some primers, in PCR buffer in case it causes double strand annealing, to see if they are visible on the gel by themselves (I'm not sure that single stranded couldn't be visible in some conformation).

[cdmatheson]

Do you have real time end point PCR capabilities? You could do a melting curve with CYBR green. This would deifferentiate between primer dimer and other templates.

[relaxin]

If raising the annealing temperature does not help, you can use a polymerase (inhibited by specific antibody) that requires activation during denaturation step.