7) Design your protocol for your site-directed mutagenesis PCR reaction. The Qui

Question

7) Design your protocol for your site-directed mutagenesis PCR reaction. The QuikChange kit provides this outline: X 1 of 10x reaction buffer X | (10 ng) of dsDNA template X11 (125 ng) of oligonucleotide primer #1 X11 (125 ng) of oligonucleotide primer #2 1 ul of dNTP mix 3 ul of QuikSolution ddH2O to a final volume of 50 ! where X is unknown. Calculate X for all unknowns. This addition information is also needed: You have a stock solution if dsDNA template at 5ng/ul You have stock solutions of both forward and reverse primers at 50 ng/ul.

Explanation / Answer

Ans. #1. Given, final volume to be made upto = 50.0 uL

Using C1V1 (stock buffer) = C2V2 (working reaction mixture)

            Or, 10X x V1 = 1X x 50.0 uL

            Or, V1 = (1X x 50.0 uL) / 10X

            Hence, V1 = 5.0 uL

Hence, required volume of 10X buffer = 5.0 uL

#2. Required volume of dsDNA template = Required amount of DNA / stock [DNA]

                                                = 10.0 ng / (5.0 ng/ uL)

                                                = 2.0 uL

#3. Required volume of primer #1 = Required amount of DNA / stock [primer]

                                                = 125.0 ng / (50.0 ng/ uL)

                                                = 2.5 uL

#4. Required volume of primer #2 = Required amount of DNA / stock [primer]

                                                = 125.0 ng / (50.0 ng/ uL)

                                                = 2.5 uL

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