PCR Protocol

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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.
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