PCR Protocol

1. First create a reaction master mix on ICE, which is as follows:

These are both 1X PCR reactions

Using Expand DNA Polymerase
 * 5 μL - 10x ThermoPol Buffer
 * 5 μL - 20 mM MgCl2
 * 5 μL - 2 mM dNTP mix
 * 2 μL - 10 μM sense primer
 * 2 μL - 10 μM antisense primer
 * 0.5 μL - template DNA (~100 ng/μL)
 * 0.5 μL - Expand DNA polymerase
 * 30 μL - ddH2O

Using Phusion DNA Polymerase
 * 10 μL - HighFidelity Buffer
 * 5 μL - 2 mM dNTP mix
 * 2.5 μL - 10 μM sense primer
 * 2.5 μL - 10 μM antisense primer
 * 0.5 μL - template DNA (~100 ng/μL)
 * 0.5 μL - Phusion DNA polymerase
 * 29 μL - ddH2O

2. Transfer the PCR reaction to a PRE-HEATED block, so that primers do not misanneal. The cycling program is as follows:

Initial 2 minutes at 94°C for Expand DNA polymerase or 98°C for Phusion DNA polymerase Cycle: 20-30 times
 * 0.5-1 minute at 94°C (Expand) or 98°C(Phusion)
 * 0.5-1 minute at 45-70°C (See below for how to calculate annealing temperature)
 * 1-5 minute at 72°C (See below for how to calculate elongation time)

Final 10 minutes (Expand) or 7 minutes (Phusion) at 72°C Hold at 4°C


 * a. Annealing temperature is 5°C below the melting temperature of the primers.
 * b. Elongation time is roughly about 1 minute per kb of product length.