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.