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#1 Bassaml7

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Posted 06 June 2011 - 07:57 AM

Hi,

I read that Taq should not be freezed. The commercial stock solution contains 50% glycerol so it does not freeze at -20. However a lot of suppliers sell 2X master mixes with Taq pre-added. These master mixes obviously do not contain 50% glycerol because that's inhibitory to PCR. So perhaps they freeze at -20. The question is, can I prepare my own master mix with Taq added to the mastermix and store at -20? Is it ok if Taq freezes within the master mix?

Also, what if I need to store my PCR materials for a long term? Can I store the stock solutions of dNTPs, Primers and Taq at -80 C without problems? Has anyone tried that already?

Thanks a lot.

Edited by Bassaml7, 06 June 2011 - 08:00 AM.


#2 JesseC

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Posted 07 June 2011 - 12:24 PM

Hi Bassam,
I actually do this regularly. I'll make a MM for about 20 or so rxns and then just add it to whatever sample I'm wanting to do PCR on. I keep it at 4 degrees and usually use it up within a month. I'm not sure if this is all the way it should be done but I know I haven't had a problem with it.
-Jesse

#3 Bassaml7

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Posted 08 June 2011 - 06:48 AM

Hi Bassam,
I actually do this regularly. I'll make a MM for about 20 or so rxns and then just add it to whatever sample I'm wanting to do PCR on. I keep it at 4 degrees and usually use it up within a month. I'm not sure if this is all the way it should be done but I know I haven't had a problem with it.
-Jesse

Thank you for your reply. Do you prepare your mastermix with the primers added also? I am surprised that you store the whole mix with Taq added at 4 c because this can cause primer dimer formation especially for prolonged periods of storage. Taq has a 1-5% activity at 4 C. Also dNTPs are not very stable at 4 C. Do you use the standard Taq? or some kind of highly stable Taq? In fact there are some commercially available Taq mastermixes stable at 4 C produced by many suppliers, so are you sure you're talking about the standard PCR materials?

#4 JesseC

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Posted 08 June 2011 - 07:12 AM

No I make my mastermix with just water, buffer, dntps, and taq. I add the template and primers in to each individual reaction. Like I said, I'm not sure if this is ideal and I don't use it for any sort of quantitative PCR, I just use it mainly for genotyping or cloning. I also use it up relatively quickly so I don't think the dNTP degradation becomes a big problem. And I use standard taq.

Thank you for your reply. Do you prepare your mastermix with the primers added also? I am surprised that you store the whole mix with Taq added at 4 c because this can cause primer dimer formation especially for prolonged periods of storage. Taq has a 1-5% activity at 4 C. Also dNTPs are not very stable at 4 C. Do you use the standard Taq? or some kind of highly stable Taq? In fact there are some commercially available Taq mastermixes stable at 4 C produced by many suppliers, so are you sure you're talking about the standard PCR materials?



#5 Bassaml7

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Posted 08 June 2011 - 12:24 PM

No I make my mastermix with just water, buffer, dntps, and taq. I add the template and primers in to each individual reaction. Like I said, I'm not sure if this is ideal and I don't use it for any sort of quantitative PCR, I just use it mainly for genotyping or cloning. I also use it up relatively quickly so I don't think the dNTP degradation becomes a big problem. And I use standard taq.


Thank you for your reply. Do you prepare your mastermix with the primers added also? I am surprised that you store the whole mix with Taq added at 4 c because this can cause primer dimer formation especially for prolonged periods of storage. Taq has a 1-5% activity at 4 C. Also dNTPs are not very stable at 4 C. Do you use the standard Taq? or some kind of highly stable Taq? In fact there are some commercially available Taq mastermixes stable at 4 C produced by many suppliers, so are you sure you're talking about the standard PCR materials?

I see, I think I'll try that. Thanks a lot for your help :)




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