HOW to Eliminate PCR primer dimer

Hedley Carr bmbthc at bmb.leeds.ac.uk
Tue Feb 4 09:55:13 EST 1997


In article <854842535.30333 at dejanews.com>, u8251012 at cc.kmc.edu.tw wrote:
>Dear netter:
>I have Some questions about PCR (polymerase chain rex) 
> and need your advices !
>
>1.How can I remove (or reduce) the primer dimer in PCR reaction
>   how about increase annealing Temp ? or lowering Ma2+ conc.

Try lowering the primer concentrations?
Increase template concentration?

>2.After 38 cycles, a faint signal was obtained. Can I increase 
>   cycle number, or template DNA  or even dNTP content 
>   TO further INCREASE THE SIGNAL I WANT ??

That's quite a lot of cycles! Yes, increasing template may improve your yield.
dNTP's are normally in large excess in PCR anyway so that probably won't make 
any difference.
DEcreasing the annealing temp may help.

>3.What's the best methods to elute out the PCR product with
>   high yields and low DNase contaminated. ANY kits suggested ?

Previously, I have just cut the band required out in a minimum of agarose, 
chopped it up finely on a scrupulously clean glass plate, stick in an 
eppendorf, add 100 microlitres TE8 buffer, vortex 30 seconds, and leave o/n in 
fridge at 4 degrees. In the morning, spin down 10min, full speed in microfuge 
and use approx 1 microlitre for a PCR (this may obviously vary but I have had 
excellent results even after cutting out extremely faint bands).

Hedley Carr.

Dept. Biochemistry & Mol. Biol.,
University of Leeds,
Ls2 9JT,
UK.
email: bmbthc at bmb.leeds.ac.uk




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