Thread: PCR

Hi,

I'm having real trouble amplifying a gene of interest by PCR. I have tried it several times now, and have tried adjusting the annealing temperature. By agarose gel electrophoresis i get nothing (except the DNA ladder), except once at a reduced annealing temperature I got non-specific product bands.

I'm beginning to think that maybe I am inadvertantly introducing pipetting errors. If pipetting errors are introduced into the PCR reaction, are they likely to cause the PCR reaction to fail? I guess that even a tiny pipetting error would make a big difference to the overall concentration in the reaction tubes.

Also, does it matter in what order things are added to the reaction tubes? I have been doing this:

Make a 5 x mix containing

MilliQ H2O 152.5uL (to make it up to 50ul per reaction)
Pfu buffer - 25uL at 10x
Forward primer - 25uL at 10uM
Reverse primer - 25uL at 10uM
dNTPs - 12.5uL at 10mM

(Added in this order)

48uL of this 5 x mix was pipetted into each of 4 reaction tubes

1uL template DNA was added per tube, followed finally by 1uL Pfu

The reaction tubes were spun down in a microfuge at 4.5 krpm before being put into the PCR machine

Please, if anyone could advise me as to what I am most likely to be doing wrong, I would be eternally grateful. I REALLY want this PCR to work!

Is it most likely to be a pipetting error? The wrong order of adding things?

Best wishes,

Tridimity

No, it will not be down to pipetting errors.

Either your DNA is leading to problems (try using less DNA, diluted into TE, and run some of your DNA on a gel to gauge purity and integrity) or your primers or dNTPs are no good. Double check your primer sequences and try new aliquots, also try a fresh aliquot of dNTPs.

Your order of addition to the master mix is fine.

Thanks,

I will try again

what's your positive control?

My positive control was TAB1 and primers, which worked recently on the same PCR programme for someone else in the Lab, so it is definitely something I personally am doing wrong

Still don't know what. Depressing

I have tried using fresh reagents, changing one at a time and then finally the whole lot

I think I may have added the wrong volume of H2O to the 5 x mix.. I think I was 10uL out

Have you tried using less template. Do you know that your primers are perfect matches for the sample you are targeting?

Yes, the primers are perfect

Maybe there is a primer template mismatch or the CG content is too high. Try lowering the annealing temperature.

Well, that leaves your template as the most likely suspect. Depending on your extraction protocol, any number of compounds could prohibit amplification. Phenol and chloroform are two likely suspects, and provide excellent reasoning for including a positive control in every run. Depending on the source of your material (tissue, environmental sample, etc), other chemicals such as humic compounds, which copurify with DNA, can interfere with amplification.

Any of these problems can be easily overcome by reducing the amount of template that you add to your reaction. Try a 1:10 dilution and a 1:40 dilution. Alternatively, dialyse your DNA sample to remove potential inhibitory compounds. A fresh extract is another possibility; be certain to remove all traces of phenol and chloroform or other organic solvents, which are excellent denaturants, in your next extraction.

This of course is all a long-winded way of asking whether you have tried diluting your template.

Originally Posted by tridimity

My positive control was TAB1 and primers, which worked recently on the same PCR programme for someone else in the Lab, so it is definitely something I personally am doing wrong

Still don't know what. Depressing

I have tried using fresh reagents, changing one at a time and then finally the whole lot

I think I may have added the wrong volume of H2O to the 5 x mix.. I think I was 10uL out

Let me rephrase the question.

what is your positive control for your DNA sample then?