4 replies to this topic

[Bassaml7]

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.

[JesseC]

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

[Bassaml7]

JesseC, on 07 June 2011 - 12:24 PM, said:

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?

[JesseC]

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.

Bassaml7, on 08 June 2011 - 06:48 AM, said:

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?

[Bassaml7]

JesseC, on 08 June 2011 - 07:12 AM, said:

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.

Bassaml7, on 08 June 2011 - 06:48 AM, said:

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