We want to set up a PCR reaction that that has a total volume of 50 ul. The reac

Question

We want to set up a PCR reaction that that has a total volume of 50 ul. The reaction is composed of: X ul Buffer stock solution 2 ul dNTPs mix (deoxy-nucleotide triphosphates) 1 ul Primer 1 1 ul Primer 2 X ul MgCl2 Water to 50ul 1) The stock solution of Primer 1 is 20uM. What is the final concentration of Primer 1 in the reaction if we use 1ul of the Primer 1 stock solution? 2) The stock solution of Primer 2 is 20uM. What is the final concentration of Primer 2 in the reaction if we use 1ul of the Primer 1 stock solution? 3) The final concentration of MgCl2 in the 50ul reaction is 1.5mM- how much MgCl2 should be added if the MgCl2 stock solution is 25mM? 4) The buffer stock solution is 10X- how much of the 10X buffer stock solution is added to the 50ul reaction to get a final working conc. of 1X?

Explanation / Answer

Solution: Total Volume of the reaction mixture, V1 = 50 ul

1)   Concentration of stock solution of primer 1 , M2 = 20 uM

Volume of Primer 1 stock solution added to the reaction mixture, V2 = 1 ul

Concentration of Primer 1 in the reaction mixture, M1 = ?

Using M1 x V1 = M2 x V2

           M1 x 50 ul = 20 uM x 1 ul

            M1 = 20 uM x 1ul

50 ul

            M1 = 0.4 uM

So, Concentration of Primer 1 in the reaction mixture ,M1 = 0.4 ul

2)   Concentration of stock solution of primer 2 , M2 = 20 uM

Volume of Primer 2 stock solution added to the reaction mixture, V2 = 1 ul

Concentration of Primer 2 in the reaction mixture, M1 = ?

Using M1 x V1 = M2 x V2

           M1 x 50 ul = 20 uM x 1 ul

            M1 = 20 uM x 1ul

                           50 ul

            M1 = 0.4 uM

So, Concentration of Primer 2 in the reaction mixture ,M1 = 0.4 ul

3. Concentration of stock solution of MgCl2 , M2 = 25 mM

Concentration of MgCl2 in the reaction mixture, M1 = 1.5 mM

Total volume of the reaction mixture, V1 = 50 ul

Volume of MgCl2 stock solution added to the reaction mixture, V2 = ?

Using M1 x V1 = M2 x V2

           1.5 mM x 50 ul = 25 mM x V2

            V2 = 1.5 mM x 50ul

                           25 mM

         V2 = 3 ul

So, Volume of MgCl2 stock solution that should be added to the reaction mixture = 3 ul

Related Questions

Navigate

• Browse (All)
• Browse W
• Subjects

Integrity-first tutoring: explanations and feedback only — we do not complete graded work. Learn more.