In article <854842535.30333 at dejanews.com>, u8251012 at cc.kmc.edu.tw wrote:
>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
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).
Dept. Biochemistry & Mol. Biol.,
University of Leeds,
email: bmbthc at bmb.leeds.ac.uk