Question

Prepare a PCR reaction using the following recipe.

5x buffer..............................5 μl

25 μM forward primer..........2 μl,

25 μM reverse primer..........2 μl

5 ng/μl Template..................1 μl

2.5 mM (each) dNTPs..........4 μl

Water....................................30 μl

Phusion polymerase.............1 μl

Q6. What are the final concentrations of the buffer, the forward primer, the template, and the dNTPs in the PCR reaction? Leave all concentrations in the same type of units they were provided in (x, molarity, or weight per volume). Remember the equation C1 x V1 = C2 x V2

Buffer:

Forward primer:

Template:

dNTP’s:

Answer 1

For the calculation of all desired molarity of all suede components. Uou mest provided intisl stock concentration along .

Concentration of buffer

Initial stock of primer

Initial DNTP stock concentration et

For example if your DNTPs intial stock concentration is 2mM.

M1V1= M2V2

2mM ×5 = x 55microlitre

X= 2000× 5/55

×= 181 micromolar

In similar way , we can calculate all the molar concentration as per stock concentration.