Question

12. Given the following reagents, set up a 50uL PCR reaction: 3pts

5X Fusion Buffer

10mM dNTPs

100uM Primer 1

100uM Primer 2

2000 U/ml Fusion Hi-fidelity DNA polymerase

500ng/ul plasmid DNA

Reaction conditions

200uM dNTPs

0.5uM each primer

1 unit DNA polymerase

100 ng plasmid DNA

Answer 1

You can make the PCR mixture by the following calculation.

1. 1uL of dNTP stock reagent which will contain 200uM dNTP

2. 0.25 uL of primer 1 and primer 2 each from stock reagent that will contain 0.5uM concentration.

3. o.5 uL of 2000 U/ml Fusion Hi-fidelity DNA polymerase to make 1 unit DNA polymerase

4. 5 uL of 500ng/ul plasmid DNA that will contain 100 ng plasmid DNA

5. 10 uL of 5X Fusion Buffer

5. as total it becomes 17 uL so make up the remaining volume by distilled water i.e. 33 uL of water.