Storage conditions - (Jun/06/2011 )
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.
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
JesseC on Tue Jun 7 20:24:40 2011 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?
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 Wed Jun 8 14:48:52 2011 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?
JesseC on Wed Jun 8 15:12:54 2011 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 Wed Jun 8 14:48:52 2011 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
