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taq and PCR - (Nov/18/2009 )

I changed from one company (promega) taq to another (NEB), and my PCR are not working. what could be the problem? the first taq was hotstart and I changed to a taq polymerase from a different company but not hotstart.

-Esta-

did you keep your running profile the same, or did you modify it for the new Taq?

-Ahrenhase-

I don't see why the new taq from NEB wouldnt work just because you change to NEB and used to use promega. Did you change any other conditions? You can do a manual hotstart yourself.

-jiajia1987-

Dear JiaJia,
what you mean by manual hotstart?

-adrian kohsf-

Jiajia, thats why I need help, it should have worked, but has not. I have not changed the conditions.
what do you mean by manual hotstart, and is it done with the normal taq, or is it only with those labelled as hotstart? the NEB one is not hotstart.
I am carrying out selective amplification for AFLP.

-Esta-

Esta on Nov 23 2009, 08:32 PM said:

Jiajia, thats why I need help, it should have worked, but has not. I have not changed the conditions.
what do you mean by manual hotstart, and is it done with the normal taq, or is it only with those labelled as hotstart? the NEB one is not hotstart.
I am carrying out selective amplification for AFLP.



By manual hotstart, I mean that you start your PCR running on the PCR machine (WITHOUT PUTTING YOUR SAMPLES INSIDE) until it reaches 95degcel (which is usually the first step), pause the machine, put your samples (which would be in PCR tubes and note that the samples will not contain any Taq polymerase at all) in the machine, and add your Taq polymerase one by one into each sample. After the taq polymerase has been added, you can let the machine run till the end. This reduces nonspecific amplification and is similar to hotstart Taq from companies where their Taq has an inhibitor that drops off at 95degcel so the PCR reaction can start. Manual hotstart is done with the normal taq, you can try that and see if it works.

-jiajia1987-

Dear Esta,

When you change your Taq, did you change the PCR tube as well to a different brand? this might be a culprit.

Second, Promega comes with 5X buffer, and NEB comes with 10X buffer, which you should be take note of. Also promega buffer doesn't have MgCl2 and Mgcl2 comes in different tube, but NEB buffer does pre-mixed with Mgcl2 which you can't optimise the mgcl2 yourself.

Perhaps this explains why your direct switching of PCR Taq doesn't work. You should tweak your PCR recipe.

Adrian

-adrian kohsf-