primer dimer in pcr product - cloning for a gene. analyzing pcr product shows primer dimer (Aug/09/2006 )

dear all,

i AM A NEW COMMER. I AM WORKING ON A PROJECT FOR WHICH WE DESIGNED THE PRIMERS.
WHEN AMPLIFICATION OF DNA IS DONE AND ANALYZED WE HAD A PRIMER DIMER FORMATION. AFTER REPEATING THE EXPERIMENT I GOT THE SAME PRIMER DIMER FORMATION. EVEN THE AMPLIFICATION WAS NOT GOOD.

SO PLEASE HELP ME WITH THIS PROBLEM AND SUGGESTIONS HOW TO GET RID OF THIS PROBLEM.

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is it possible to design new primers?

you can also decrease their concentration and/or accordingly increase the concentration of template...this can improve it but may not get rid of the dimers if the interaction is too strong

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what conc of primers did you use? do you make the master mix on ice? did you try hotstart PCR?

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U could increase the template concentration.

ALso try to heat the primers and template to 95 C for 5 min. before adding the rest of te PCR mixture.

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Thank you for your useful tip sir. ya, its possible to design a new primer and we will follow your idea.

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Thank you sir for your replay. i will see if i could get rid of the dimer by following your advice.

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For designing primers use PRIMER3: http://frodo.wi.mit.edu/cgi-bin/primer3/primer3_www.cgi
Here, you can chose the max. self-complementary and the max. 3` complementary of your primer pair. Try to keep them as low as possible to avoid primer dimer formation (between zero and three maybe).

Isabelle

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Try BLUNT-ENDED hairpin primers - they usually do not produce dimers

See literature to LUX primers from Invitrogen

or original paper in NAR

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If the primer dimer appears at a temperature lower than the real product you could introduce an extra temperature step to melt the primer dimer before the plate read. You would first need to look at the melt curve.

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