PCR with TAQ polymerase

[Silvia_84]

Hi all,

 

I have to run a PCR reaction and I am following a protocol suggested by the company of the TAQ polymerase I have. The protocol says to use 10 ul of 5x MyTaq Reaction Buffer and 0.25 - 1 ul of 2XTAQ polymerase. in the reaction Does it mean that I have to diluite both before using them since they are concentrated respectively 5X and 2X? Or shall I take the amounts suggested by the protocol from the concentrated solutions as provided by the company? It is not specified on the protocol..

Furthermore, I did not understand very well how I can calculate the volume if TAQ polymerase I need if the concentration is reported in Units.. e.g. If the protocol says to add 1.25 units of TAQ, at what volume does it correspond?Can you explain me a little bit the calculations I should do?

 

Thank you very much in advance

[BabcockHall]

I am not a PCR person; therefore, I will keep my response very general. One unit of any enzyme is the amount of enzyme needed to convert one micromole of substrate to product under defined conditions. It is an extensive property, and it is proportional to the number of moles or to the mass of an enzyme. The volume of TAQ polymerase to which this responds depends on the number of units per µL (this number is related to the specific activity of the polymerase, in units per milligram of protein). It might vary slightly from one lot of enzyme to the next.