PCR Lab calculations The plasmid template is at a concentration of 2.44 mg/µL Mu

ID: 166699 • Letter: P

Question

PCR Lab calculations

The plasmid template is at a concentration of 2.44 mg/µL

Mutation Information

1. 5_Mutation, MW = 12162.9, 50 µM in 364 µL H2O

2. 3_Mutation, MW = 11815.6, 50 µM in 411 µL H2O

Calculate the X value for a total volume of 50 mircoliters

5.0 µL 10X reaction buffer

X µL (25 ng) of MDH plasmid template

X µL (125 ng) oligonucleotide forward primer

X µL (125 ng) oligonucleotide reverse primer (-r)

1.0 µL dNTP mix

1.5 µL QuikSolution reagent

1.5 µL DMSO

X µL milliQ H2O for final volume of 50 µL (calculate amount needed)

Concentration of plasmid template is 2.44 mg/µL

For PCR reaction we need 25ng of plasmid

1mg = 106ng

So we need 25/2.4*106

1.04*10-5 µL plasmid

Given the stock solution of primer is 50 µM

We need 125 ng of each primer for PCR reaction so volume of primer needed for each reaction will be

125/50000

=1/400 = 0.0025 µL

Reaction mix will be

5.0 µL 10X reaction buffer µL

1.04*10-5 µL (25 ng) of MDH plasmid template

0.0025 µL (125 ng) oligonucleotide forward primer

0.0025 µL (125 ng) oligonucleotide reverse primer (-r)

1.0 µL dNTP mix

1.5 µL QuikSolution reagent

1.5 µL DMSO

41 µL milliQ H2O for final volume of 50 µL

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