The PCR reactions consist of primers, cell suspension, and PCR Master-Mix at a f

Question

The PCR reactions consist of primers, cell suspension, and PCR Master-Mix at a final volume of 50 mu l. The Master-Mix contains PCR buffer, dNTPs, Taq polymerase and water. Each of the 50 mu l reactions requires 1 X PCR buffer, 0.2 mM dNTPs, and 1.25 Units of Taq polymerase. Given the following stock concentrations: 10X PCR buffer, 10 mM dNTPs, and 5 Units/mu l Taq polymerase, determine how you would prepare just enough of the Master-Mix for the six PCR reactions performed in your experiment. Water is used as the solvent in preparing the Master-Mix.

Explanation / Answer

This can be calculated by using simple molarity equation M1V1 = M2V2

Taking one of the components PCR buffer, 10 X x = 1 X 50, where x is the volume to be found out for 50ul. x = 5ul. Similarly dNTPs will come out to be 1ul and Taq will be 1.25 ul. Since there are six reactions to be performed, all the values should be multiplied by six. Hence the final volume would be PCR buffer = 30ul, dNTPs = 6ul and Taq enzyme = 7.5ul. The total volume of the master mix should be 300ul, hence milli-Q water should be used to equally adjust the volume.

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