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Tac polymerase denatures at room temp why? - PCR-tac polymerase (Nov/09/2006 )

hi every body,

here i have a doubt, Tac polymerase will be exposed to different ranges of temp’s in PCR machine more than 1hr, there it wouldn’t denatures . but it should not kept for more time at room temp (there it should be kept in ice) why so ?

-Lalitha Devi-

oh, your taq doesn't live forever at 55-95. it will denature during the course of longer pcr's, especially with long template

if you do a lot of PCR, you pull that taq out of the freezer on many many occasions, you will reduce the longevity of the enzyme for later pcr's. does that make sense? think of it like half-life for the enzyme

when you are setting the pcr up, it is also good for the reagents to be cold because taq has some activity at lower temperatures. it can chew up your oligos, cause non-specific product formation, and other bad things (look up why hot-start pcr is a good thing; also temperature requirements of some newer polymerases are more lax and their proofreading functions are varied also)


Also: the polymerase itself is kept in 50% glycerol, not in the final reaction buffer... (before making your PCR mixes). Stability in glycerol at room temp is not too good I believe (if not: why bother storing your enzyme at -20°C?).


Taq is stable for 6 months at room temperature. It is stored at -20C to prevent potential bacteria and mold contaminants floating around your lab from growing in the tube.


It's also so that when you make up your PCR mix the temperature is kept low so as to avoid mis priming which easily happens at RT

-John Buckels-