49 replies to this topic

[hobglobin]

Sunflower_gl, on 03 May 2012 - 05:35 AM, said:

Hi there,
I can't figure out my problem with my PCR. I've tried to change Mg2+ concentration, annealing temperature... but nothing happen. I have always smear bands. my lab-mate (who has done many PCR) suggested me use the Q-solution to increase PCR specificity. But i have no experience with this solution. In fact, I am doing PCR with 4mM Mg2+ concentration and i don't know if I have to decrease the concentration of Mg2+ when I add Q-solution. Do you guys have any advices?
Thanks

Q-Solution is for GC rich templates, so if it's the case it may help you, though not sure if it helps to increase specificity (usually it helps to get amplification at all in such "difficult" templates)....4 mM Mg²⁺ is a quite high concentration, I also would try to reduce it to 2 or 1.5 mM (and sometimes even less concentrations work) as this increases specificity too a lot...
There are many other possibilities to increase specificity...but depends what you already did...

[Sunflower_gl]

I've done a temperature gradient and Mg2+ concentration gradient (from 1,5 mM to 4 mM). It appeared only some "smear" bands at 4 mM Mg2+ and nothing in the other concentrations of Mg2+. So I supposed that Mg2+ had some effects on its specificity but it wasn't enough. Here are sequences of my reverse and forward primers:
agc ttg aac cac cag ggt ata cgt agg
tct aca gca tca ctg gcc aag gag ctg

What do you think about that? If I add Q-solution to the mix, do I have to decrease the Mg2+ concentration?
Thanks

[memari]

We have used this and now we have 5 fold more DNA for QPCR for Chip. It is definitly better tha QIAGEN.
Feldan's PCR Purification Kit
http://www.feldan.co...on-kit-200preps


Babak

[hobglobin]

Sunflower_gl, on 09 May 2012 - 12:19 AM, said:

I've done a temperature gradient and Mg2+ concentration gradient (from 1,5 mM to 4 mM). It appeared only some "smear" bands at 4 mM Mg2+ and nothing in the other concentrations of Mg2+. So I supposed that Mg2+ had some effects on its specificity but it wasn't enough. Here are sequences of my reverse and forward primers:
agc ttg aac cac cag ggt ata cgt agg
tct aca gca tca ctg gcc aag gag ctg

What do you think about that? If I add Q-solution to the mix, do I have to decrease the Mg2+ concentration?
Thanks

First I'd keep the Mg²⁺ concentration as it was (4 mM), otherwise you don't know if the Q-solution or the changed Mg²⁺ concentration was the reason for fail/not fail. Anyway other reasons also might be a reason (DNA quantity or quality, inhibitors in mix, primers just don't work for the template (designed how?), other PCR ingredients are dead (positive control working?), PCR program sucks,...).
You also can try out other polymerases, sometimes they just work for whatever reason....The primers look good as far as I can judge it.

[Sunflower_gl]

Thanks Babak and hobglobin.

I've always done PCR with at least one positive control and it worked very well. That's why i don't understand the reason of those "smear" bands! In the first time, I'll add Q-solution and conserve the concentration of Mg2+. Let's see how it works and I'll show you my result soon!

Sunflower_gl