Team:British Columbia/Notebook/Protocols/PhusionPCR

UBC iGEM 2015

Colony PCR Protocol for Taq DNA Polymerase with Standard Taq Buffer

Overview

PCR 

The Polymerase Chain Reaction (PCR) is a powerful and sensitive technique for DNA amplification (1). Taq DNA Polymerase is an enzyme widely used in PCR (2). The following guidelines are provided to ensure successful PCR using NEB's Taq DNA Polymerase. These guidelines cover routine PCR. Amplification of templates with high GC content, high secondary structure, low template concentrations, or amplicons greater than 5 kb may require further optimization.

Protocol

Reaction setup: 

Assemble all reaction components on ice and quickly transfer the reactions to a thermocycler preheated to the denaturation temperature (95°C). 

Component 10 µl Reaction 50 µl Reaction Final Concentration
10X Standard Taq Reaction Buffer 1 µl 5 µl 1X
10 mM dNTPs 0.2 µl 1 µl 200 µM
10 µM Forward Primer 0.5 µl 2.5 µl 0.5 µM
10 µM Reverse Primer 0.5 µl 2.5 µl 0.5 µM
10 µM Forward Primer Larry the Bird @twitter
10 µM Forward Primer Larry the Bird @twitter
10 µM Forward Primer Larry the Bird @twitter