PCR Amplification

PCR Protocol
Materials:

Primers (FWD, RVS)

PCR Supermix

Parent plasmid

Methods:

1.	Measure the concentration of the primers and the parent plasmid as follow: Do a dilution of 50:1 with 1EB buffer [ do 98ul tris into 2 ul of DNA ] Use the below formula to find the concentrations

Conc. (ng/ul) = OD reading * dilution factor * DNA factor = OD reading * 50 * 30

2.	Mix 90ul of PCR supermix with 80ng of each DNA, 200nM of each primer

3.	Put 90µl in 0.5ml tubes.

4.	Find the length of the PCR product from Vector NTI

5.	Run the PCR program

Thermocycler program:

Step1: 95C for 5 min.

Step2: 95C for 15 sec.

Step3: [lowest primer annealing temperature] for 60 sec.

Step4: 68C for [1 minute + 1 minute per 1 kb).

Go to Step 2: 35 times.

Step6: Hold at 4C.