Would anybody would like to share how commercial PCR mixes manage such long storage lives? Given that the Taq is already a part of the mix, it must be quite difficult to maintain its integrity through frequent freeze thaw cycles. Is it just glycerol that gives it a longer shelf life?
hi
Enzymes are usually sensitive to freezing and Thaw., Even ATP molecules On the basis of these products are also susceptible to this condition.The best way to keep this kind of segmentation products when you buy them in smaller quantities (in size each time it is needed) and keep the -20 c freezer.Taq DNA Polymerase Master Mix Red for Ampliqon company is ok.
good luck
Dear Paleja, For routine use vial, i use to store at 4 degree as by instruction of manufacturer (Emerald Takara). they should be stored at -20 degree until u started to first use. Once u star using, avoid to store at -20 degree again because freeze - thaw cause loss of activity. Also should be noted that your Ready made PCR mix should contain Hot start Taq.
Dear all,
Thank you for your replies.
I am aware that PCR mixes should be stored at -20 degrees for long term storage.
What I would like to know is what are the ingredients the PCR mix that maintain the integrity of the polymerase even after long shelf storage.
Ameya
New generation recombinant Taq DNA polymerase are complexed with a proprietary antibody that inhibits polymerase activity at normal temperatures. Polymerase get activated only when temperature reaches 94 deg Celsius.
Hi Pranav,
Thanks for your input. What do you reckon is added to the polymerase's storage buffer for enhanced integrity?
DMSO and glycerol are both good candidates for cryoprotection - and are often components of Taq mastermixes. Many (if not most) formulations are proprietary, so these are guesses, but, the abilities of DMSO and glycerol to prevent the full tetrahedral lattice crystallization of water is relatively well-known. In addition, Taq is actually stable for years at room temperature - so it is pretty tough over all. DMSO and glycerol (in addition to being cryoprotective) serve as PCR enhancers in various regards as well...
There is some missing information in the question. There is a difference between shelf life and protection from freeze-thaw. We've had mixes in our -20 for over 18 months, with no adverse effects (when comparing control reactions). On the other hand, some show differences after only a few months if there are many freeze thaw cycles. What type of preservation are you looking for?
Dear Jack,
Thank you for your reply.themes quite useful.
Mark,
Yes, probably there is some ambiguity in the question. Basically I want to know what protects the enzyme from multiple freeze thaw cycles. From what Jack said, something like Taq is strong by itself. Is there something different that one should do if we are looking to store Bst polymerase and protect it from frequent freeze thaws?
Hi I have seen people using Trehalose, 0.1 mM EDTA, 1 mM DTT, Tween 20 and 0.5% Nonidet-P 40 mix as protectant.
hi Ameya
had you tryied which Pranav recomended, had ever obtained any tesult.
Thanx
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