Hello,
Did the producer recommend keeping the master mix in the fridge? You can find this detail written on the box or . Maybe the room temperature conditions will affect the pcr results
Hi Shafia,
I'm afraid since your mix contains the taq (enzymes are sensible to temperature) that it is damaged, but we do not know. you can test it on a PCR you know is performing well.
fred
Hi Shafia,
Our MyTaq HS is very stable (we test it at -20oC and +4oC for extended times), we have stabilizers in the buffer, however at higher temparatures you can get damage to the polymerase and antibodies. We therefore cannot guarentee the quality if left at room temperature. I would definately recommend a new box, but run a PCR using the old on in parallel to see how it performs.
Hi Shafia...
Definately this will affect your Mastermix, but to which extent?? I will go with Fred suggestion, experience it, then make your call based on the result!
Regards
Hi all,
Maybe my question is idiot but if there is no DNA polymers inside the mix (template or primer), the Taq and the others components are supposed to work at 95°C, so what could happen below ? Maybe dNTP will be corrupted ?
First Of all you must know the condition of storing your product.
second thing you can tell us what is the weather condition in this 24 hrs (i just try to know the conditions of weather in your lab).
most of PCR master mix can tolerate 4 C for some of time.
if you dont know the condition of weather as temperature and humidity in your lab at this 24 hrs which your PCR master mix was outside the storing unite the only way to examine the vitality of this chemicals by running PCR reaction with positive sample using primer that you confirm it activity.
N.B : PCR master mix contains (Taq Enzyme, Nitrogenous bases, Co factors for the reaction)
all of these components can tolerate the temperature till 15 C for 3 days
as
1- Taq enzyme works at temperature over 70 C
2- Nitrogenous bases tolerate room temperature 15 C for 3 dayes
3- Co Factors as buffers and some chemicals eg. Mg Cl, Na Cl, Na Ac all these chemicals not damaged by room temperature .
So you must firstly to confirm the room temperature that your Master mix was in in these 24 hrs and after that you can determine your decesion if you continue with this master mix or buy anew one.
I will suggest u to check it first for 10 microlitre of ur PCR rxn if there is a perfect band, then u can use.
All the very best.
Hi Shafia,
I have had similar situations before. Depending on the weather conditions and temperature, your Master Mix will be functional to some extent. Although the enzyme can stay stable at temperatures as high as 95C during denaturing step, long exposure to high temperatures will degrade nucleotides and reduce enzyme activity. However, if your lab is air-conditioned well, there is a good chance it will still be fine. As other people have recommended, it's better to test it with a known template.
I am attaching a result of PCR that I did with NEB Phusion 2xMMix that had been left out on the bench for one day (~24 hours) and the normal one (new aliquot).With the old aliquot the band is barely visible. In my case, the enzyme had lost activity almost completely.
I agree with other contributors that it doesn't really matter at the global laboratory temperature range which in most cases should not reach beyond 40oC
Hi Dear Shafia Zahra ,
I experienced this situation two years ago, but as Frederic Lepretre and others said, enzymes are sensible to temperature, although in my case there was no problem and I used that MasterMix in my project and no problem was observed.
I think it depends on which company produced it.
If you have ample amount of enzymes, primers and etc, you should check it.
Good luck
Kheirvari JK
You can test if the master mix is working doing a PCR Reaction.
I am assuming that the master mix in question contains polymerase. Thus, run 2 controls - 1) empty vector (no insert) and 2) no DNA.
Hi
You can test it using negative and positive controls of a known PCR reaction.
Depends on your sample volume. If you can use it on a very very small sample, keep the rest of the sample safe. Else use it on a known control to validate whether its reliability is compromised. If its not compromised, it may be used for the test sample.
For sure it is a good idea to test in a positive control reaction or against another batch you might have in the lab. The polymerase will loose activity when exposed to long time room temperature. To answer whether you can use it or not depends largely on your application. If you want to clone a large fragment by PCR it would be for sure no. If you want to do a simple genotyping etc. it might be good enough.
If it contains Tag enzyme you can not otherwise, it is ok for an overnight, no problem.
Just run a +ve and -ve PCR reaction using one of your housekeeping gene, in case you are dealing with plasmid. use any pUC control vector from any plasmid prep you have with the right primers.
No need to buy new one first you just check its working or not. Then take final decision after confirmation...
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