PCR

Specific genes are amplified from genomic DNA with the polymerase chain reaction (PCR) performed in a thermocycler. Since the PCR conditions for gene amplification can strongly vary according to the nature of the template DNA and the targeted genes, a specific protocol must be checked and modified if necessary. In principle, all parameters can be modified (e.g., primers, amounts of the various reagents, type of the Taq DNA polymerase, numbers of reaction cycles, temperature of single cycle steps, etc.) and the best result is open to tests. The following protocol was successfully used as a standard protocol for the amplification of 16S rDNA:

Reagents and solutions:

10 x Taq-Reaction buffer (TaKaRA, Shiga, Japan)

10 x dNTP solution (Roche, Mannheim, Germany) 2.5 mM dATP 2.5 mM dCTP 2.5 mM dGTP 2.5 mM dTTP

10 x BSA solution (bovine serum albumine): 3 mg/ml in PCR water

F = forward primer, R = reverse primer
M = A/C, R = A/G, Y = C/T

(Continue with PCR methods on next page)



back to top