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PCR Amplification
From 2008.igem.org
PRINCETON IGEM 2008
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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.
