Team:Michigan/Notebook/PCRProtocol

PCR Standard Protocol

PFU turbo enzyme or Taq polymerase were used as enzymes. Taq is cheaper, but error prone, so only used for side screening.

1. Determine the total volume you wish to use - usually 50 or 100 μL
2. Add 1 μL of your DNA vector for every 50 μL total volume to PCR tube
3. Add 1 μL of each primer for every 50 μL total volume
4. Add 5 μL of dNTP for every 50 μL total volume
5. Add 5 μL of the correct buffer solution (depending on enzyme used) for every 50 μL total volume
6a. If using PFU, use 1 μL for every 50 μL total volume, and the balance deionized water (add enzyme last)
6b. If using Taw, use 0.5 μL for every 50 μL total volume, 1.5 μL of MgCl, and the balance deionized water (add enzyme last)
7. Follow protocol for running PCR machine - This will depend on the type of PCR machine used. Be aware that some PCR machines may also require different mixtures to run properly. This is the protocol specified for our machine, the PCR Express.

Following this protocol, your PCR mixtures should look similar to the following:

EXAMPLE PCR 1: 50 μL total volume using Taq polymerase
35 μL dd water
5 μL dNTP
5 μL Taq buffer
1 μL Primer 1
1 μL Primer 2
1 μL DNA vector
1.5 μL MgCl
0.5 μL Taq polymerase

EXAMPLE PCR 2: 100 μL total volume using PFU turbo
72 μL dd water
10 μL dNTP
10 μL PFU buffer
2 μL Primer 1
2 μL Primer 2
2 μL DNA vector
2 μL PFU turbo