Why is it important to use ddH2O in your Master Mix, rather than dH2O or tap wat

Question

Why is it important to use ddH2O in your Master Mix, rather than dH2O or tap water?

What is the purpose of positive and negative amplification controls?

How is a negative amplification control different from an extraction reagent blank?

Why should your Master Mix be kept on ice at all times? Is this absolutely necessary for reactions utilizing “hot-start” polymerases? Was the polymerase used today a hot-start polymerase?

Several temperatures were used during the thermal cycling program for your D1S80 amplifications. Describe what is happening in the tube during the 94°C / 90°C, 64°C, and 70°C temperature settings. What is the purpose of the 4°C hold step at the end of the program?

Explanation / Answer

Nuclease free water is used in order to dilute the concentration of the reagents to the proper final concentration. Also use of nuclease free water helps avoid DNA degradation by nucleases as well as interference of the PCR reaction by ions which could be present in otherwise not nuclease free deionized water.

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