VPB 321: Animal Biotechnology (2+1)

POLYMERASE CHAIN REACTION

Aim: To demonstrate Polymerase chain reaction.

Materials required:PCR primers, 10 X PCR buffer (Tris-HCl, KCl, MgCl₂), dNTPs, Taq DNA polymerase, PCR tubes, Micropipettes and Thermal cycler.

Method

• Centrifuge the culture samples (2ml) at 10,00 rpm for 1 min.
• Resuspend the pellet in 0.5 ml of TE Buffer and wash two or more times with minimum amount of distilled water.
• Resuspend the final pellet in 100 u l of TE Buffer. Use 5 u l of this sample for PCR assay.
• Heat the samples to 96 ° C for 10 min for denaturation and keep in ice.
• Perform amplification of DNA in a total volume of 50 u l.
• The reaction mixture contained 5 u l of 10X PCR reaction buffer (50 mM KCl, 20mM MgCl_2, 10mM Tris-HCl, pH 8.3).
• Primers used at a concentration of 100 m M. The four dNTPs (dATP, dGTP, dTTP and dCTP) used at a final concentration of 10 mM each.
• 1 ul of Taq Polymerase added.
• Amplify in a Thermal cycler for 32 cycles, each cycle consisting of denaturation of DNA for DNA for 2 min at 94 ° C, annealing of the primers for 1 min at 55 ° C and elongation for 2 min at 72 ° C.
• Analyze the PCR products by gel electrophoresis on a 1.5 % agarose in Tris AE borate buffer, pH 8.3.

Questions

• Define Taq DNA polymerase.
• Write the properties of Taq DNA polymerase.
• Write the advantages of PCR technique.
• Write the role of PCR buffer in PCR.
• Name different methods of PCR.
• Write the applications of PCR.