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Summary: PCR Amplification
1. PCR Reaction:
2.5 µl 10X Taq polymerase buffer
2.5 µl 10X dNTP mixture (2.5 mM each dNTP, 10 mM total dNTPs, store at -20°C)
1 µl Primer 1 (12.5 µM = 12.5 pmol/µl) (final concentration = 0.5 µM = 0.5 pmol/µl)
1 µl Primer 2
1 µl Template DNA (0.01-1 ng/µl, use 25 ng gDNA, use 0.25 ng plasmid DNA)
17 µl Sterile dH2O
25 µl Total volume
0.25 µl Taq polymerase (5 units/µl)
10X Taq buffer: 200 mM Tris-Cl, pH 8.4 (at 25°C) (filter sterilize, store frozen)
500 mM KCl
15 mM MgCl2
dNTPs: Store 100 mM dNTP stocks at -80°C. Combine equal volumes of 100 mM stocks to
prepare a 100X stock that is 25 mM for each dNTP. Prepare 10 µl aliquots of the 100X stock.
Dilute an aliquot ten-fold with sterile dH2O to prepare 10X stock that is 2.5 mM for each dNTP.
2. Thermal Cycling:
Initial denaturation = 94°C for 2 minutes
Denaturation = 94°C for 30 seconds
Annealing temperature = 45 - 55°C for 30 seconds (Tm 5°C)
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