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Could higher ammount of taq be inhibitory for overall PCR reaction? - (Feb/19/2006 )

some of my pcr products are showing no amplification of desired product with higher amount of taq ie equal to or more then 2.5U in 25 micro liter reaction mix. the intensity of band decreases with the increase in the amount of taq, so is the case with the lower amount of taq.

i have not yet came across any articles mentioning the same so i was wondering can it the due to higher quantity of taq or any other reason behind it.

-sanjiv kumar-

It may not be the Taq per se, but what the Taq is stored in...

Similar to restriction enzymes, where you do not want the enzyme volume added to exceed 1/10 of your total reaction volume, because restriction enzymes are usually stored in 50% glycerol and if you exceed 5% glycerol in your final reation mix, the digestion will be inhibited.

Taq is a very hardy enzyme (which is why it works in PCR); you need very little of it for the reaction to succeed. It is also quite expensive.

Why are you using so much?

-HomeBrew-

QUOTE (HomeBrew @ Feb 19 2006, 09:21 PM)
It may not be the Taq per se, but what the Taq is stored in...

Similar to restriction enzymes, where you do not want the enzyme volume added to exceed 1/10 of your total reaction volume, because restriction enzymes are usually stored in 50% glycerol and if you exceed 5% glycerol in your final reation mix, the digestion will be inhibited.

Taq is a very hardy enzyme (which is why it works in PCR); you need very little of it for the reaction to succeed. It is also quite expensive.

Why are you using so much?



i am just tryin to standerdize the pcr for brucella in department and it is not that common here so i am tryin to explore all the possibilities for its standerdization and i got those results, one of my enzymes is not givin satisfactory results at higher concentration.

-sanjiv kumar-