PCR primer dimer? - (Jun/15/2005 )
Hi,
I am new to PCR. When I run my gel I get a faintish bands for specific products and get a thick band at the end of the run (low molcular). Is it due the reamining of nucleotides?
What is the remedy?
My control band appear ok
Viskam
u may try with standardizing with various mgcl2 concentration of the pcr to increase the specifity or else u can also use DMSO or Glycerol etc., to increase the sepcificity if u feel that the pcr program is ok or else u try optimising the annealing temperature of the pcr
good luck
I think the bright band at the bottom of the gel may be primer dimers. If your target band is good, then just reduce the pimer concentration. If your target band is fainted, try to reduce annealing temp. You can check with various softwares for your primer sequences, if dimer exist, better redesign it, if redesign is impossible, then try some additives as suggested above. Do not increase the Mg concentration too high as you may get unspecific bands.
Cheers!
dtle, can you suggest some programs(preferebly online) to check primer dimer formation?
Hi Viskam,
I think parimacpbg is correct, that you primer dimer.
There are several softwares to use:
1) FastPCR (freeware, you can download, just google it.....)
2) AnnHyb.......
These program can predict Tm of primer dimer, let say 42oC, so you have to set you annealling temperature higher then 42oC to prevent primer dimer formation.
Also, check your 3' end of your primer, make sure there is no:
i) over running of A/G/C/T for more than 4, particully for G.
ii) no palindromic sequence at 3' end.
Good luck
Hadrian
I have used intensively the NET PRIMER (Online, java applet)
Why should we increase the annealling temperature to prevent primer dimer?
Hi indoubt,
As the primers are not directly complementary they will not anneal together as strongly as they will bind to the sequence to be amplified. therefore if you raise the annealing temperature, while maintaining it below the annealing temp of binding to the specific amplicon then you should be able to at least reduce primer dimer.
Scott