PCR stopped working

[pedronog]

Hello everyone,

I'm going crazy with this...

I have a Taq from Biotools that comes with buffer with MgCl2, buffer without MgCl2 and MgCl2 solution.
I used to perform colony PCRs almost everyday and it always worked with the provided buffer with MgCl2. Always getting clean bands.
Untill one day when buffer with MgCl2 finished. I replaced this buffer with the buffer without MgCl2 and added MgCl2 separately so the reactions could have the same concentration of MgCl2 as the one provided already with MgCl2 (2mM).

The only thing that changed was the buffer and it just stopped working.
When I run the agarose gels, in the reactions from colonyPCR I get a huge amount of dna that does not go far from the well and the controls show a strong band with a shorter size than expected (~750bp). It seems that the expected band is there (~1.000-1.200bp) but it's too weak... Some smearing can also be seen... Negative controls (without dna) are 100% clean.

what's happening???!

Thanks

[jonoave]

Sorry to hear bout your problems. However, these things tend to happen, sometimes without rhyme or reason. ;P

I'm actually quite surprised that you can finish your buffer before the Taq, considering that the manufacturer usually provides a surplus of them. For the Taq I use, I don't even finish the first tube of buffer, and they usually give 2 tubes!

I assume your calculations of the concentrations are correct. Please check the original concentration of the Mg solution (usually it's in 25 mM or 50 mM), and you also need to factor the total reaction volume of your solution. Please also remember that the Mg solution needs to be thawed properly to minimise Mg "gradient" where the concentration is uneven due to some parts being frozen/sticking to the tube.

Anyway, the only thing I'd suggest is to make sure that the Mg solution is to start from scratch. Carry out an Mg gradient, probably from 1.5 - 3 mM. You might also need to do a temperature gradient if needed.

[pedronog]

Originally Posted by jonoave

Sorry to hear bout your problems. However, these things tend to happen, sometimes without rhyme or reason. ;P

I'm actually quite surprised that you can finish your buffer before the Taq, considering that the manufacturer usually provides a surplus of them. For the Taq I use, I don't even finish the first tube of buffer, and they usually give 2 tubes!

I assume your calculations of the concentrations are correct. Please check the original concentration of the Mg solution (usually it's in 25 mM or 50 mM), and you also need to factor the total reaction volume of your solution. Please also remember that the Mg solution needs to be thawed properly to minimise Mg "gradient" where the concentration is uneven due to some parts being frozen/sticking to the tube.

Anyway, the only thing I'd suggest is to make sure that the Mg solution is to start from scratch. Carry out an Mg gradient, probably from 1.5 - 3 mM. You might also need to do a temperature gradient if needed.

Thanks jonoave.

The calculations are correct. I dilute MgCl2 25times (50mM>2mM).

This taq comes with 4 tubes of buffer, but only one of them had MgCl2 incorporated.

Maybe the problem is that i don't mix MgCl2 solution that well, just like you said... there's a warning in the tube saying that it has to be vortexed for a few seconds. I didn't give much importance to that and just shook it roughly with the fingers... Maybe that's the key for the problem!

I'll repeat it making sure it gets vortexed vigorously and then post the results.

Thanks again.

[pedronog]

Hello again...

I found that mixing better the MgCl2 solution was not the problem...

Then I tried changing everything... changing buffers, mgcl2 solution, dNTPs, primers, H20,... and nothing. Taq didn't amplify correctly...

So I tried another polymerase, in this case pfu, and it does amplify correctly... So... the problem is Taq!!!!

Now my question is... what reasons can explain why this Taq stopped working from night to day???!

Thanks again.

[luisillo]

Unproper storing (above -20ºC), too much time out of the freezer, UV irradiation of the Taq tube, contamination (most likely bacterial). I think those would be the stronger candidates.

Good luck in your experiment.

[pedronog]

yeah... thanks luisillo... I imagined those situations... i guess i had to aliquot a fraction of the taq solution for myself before... it's used a lot of times by a lot of people...

I would bet for "too much time out of the freezer" or better "too much times out/in of the freezer (we take it out of the freezer on a frozen container, but...) or "contamination" (we manipulate it on a pcr chamber previously irradiated with UV... but we never know….)...

We'll buy another taq and aliquot it in small amounts before using it...

Cheers.

[davidflora]

[Only registered users see links. ] If it gives perfect bands with other primers (specially house keeping primers) than it may be partial degradation of your cDNA b'coz of any contamination. your gene of interest may be in low amount and a slight degradation may do so. so plz do a pcr with freshly prepared cDNA from freshly isolated RNA of a good quality,.