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PCR stopped working


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#1 pedronog

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Posted 23 February 2011 - 07:39 AM

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.

Posted Image
1 marker
2 negative control
3 control 1
4 control 2
5 control 3
6 control 4
7 control 5
8 colony PCR

what's happening???!

Thanks

#2 Maddie

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Posted 23 February 2011 - 09:27 AM

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.

Posted Image
1 marker
2 negative control
3 control 1
4 control 2
5 control 3
6 control 4
7 control 5
8 colony PCR

what's happening???!

Thanks



I remember a company Tech once told me that it was important to vortex the magnesium quite a lot before the first use. I always vortex it for 7-15 seconds. I would try again. And if you want, you could also test out several MgCl2 concentrations (1.5 to 3mM).
Theory is when we know everything and nothing is working. Practice is when everything is working and nobody knows why. Here, we combine theory and practice. Nothing is working and nobody knows why.

A. Einstein

#3 pedronog

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Posted 24 February 2011 - 01:48 AM

I remember a company Tech once told me that it was important to vortex the magnesium quite a lot before the first use. I always vortex it for 7-15 seconds. I would try again. And if you want, you could also test out several MgCl2 concentrations (1.5 to 3mM).



Maybe that's the key for this problem. In the Mg tube there's a warning saying 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.

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

Thanks Maddie.

#4 pedronog

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Posted 18 March 2011 - 08:05 AM

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.

#5 Moebius

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Posted 18 March 2011 - 09:39 AM

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.


Hi. Pedronog. I am having a very similar problem. Can Pfu replace Taq and work in all PCR reactions? I thought Pfu was only used when we need to have the sample sent for sequencing?

#6 pedronog

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Posted 21 March 2011 - 03:46 AM

Hi. Pedronog. I am having a very similar problem. Can Pfu replace Taq and work in all PCR reactions? I thought Pfu was only used when we need to have the sample sent for sequencing?


What kind of problem are you experiencing?

The most important difference between the two polymerases is that pfu is much more accurate than taq. pfu has a proofreading activity and can be used for example to generate PCR inserts to further cloning because there will be a much lower probability of having an error when compared to taq.

But if you just want to check if your cloning went fine (just like in my case) you can use taq because you don't need accuracy... you'll just check the fragment size... you can also use pfu, but it's a waste of money too because it's more expensive.

Cheers




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