The definition of 1 unit is "The amount of enzyme that will incorporate 10 nmol of dNTP into acid-insoluble material in 30 minutes at 75°C." If I have to perform a unit assay does my enzyme has to incorporate 10nmol of dNTP as well? what if it incorporates 15nmol or less than 10nmol. My point is whether 10nmol is fixed or defined by the performer.
Lastly, how do I know how much nmol has been incorporated any reference for a detailed explanation of the procedure?
Hi Xin,
The definition of a unit of Taq activity is just what it is, a definition. If you do the reaction and get 15 nmol incorporated into acid-insoluble material in 30 minutes at 75°C than you have 1.5 units in your mixture. So the 10 nmol is fixed, it was defined at some point in time and now its a fixed definition of a Taq unit of activity.
I am sure there are many ways one can measure the disappearance of nucleotides by incorporation in a nucleotide polymer.
Here are some:
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3413125/
http://www.sciencedirect.com/science/article/pii/S0003269700949030
http://www.biotechniques.com/multimedia/archive/00009/02334pf02_9872a.pdf
All the best,
Milan
Hi, the unit definition you mention is completely arbitrary. The scientific definition of unit is the katal. One katal (or one unit of enzyme) catalyzes the conversion of 1 mol/s of its substrate to the product (see the link attached).
For Taq various unit definitions are in use because of the high commercial value of the enzyme (especially when it was still patented!) and these are still being used today. In general, in molecular biology, many "creative" unit definitions are in use. So, for your case, Milan's description is correct.
http://onlinelibrary.wiley.com/doi/10.1111/j.1432-1033.1979.tb13116.x/pdf
Hi, when uploading the link, it is truncated. The complete link reads as follows: http://onlinelibraray.wiley.com/doi/10.1111/j.1432-1033.1979.tb13116.x/pdf
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