Team:Michigan/Notebook/PCRProtocol

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 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 &mu;L 2. Add 1 &mu;L of your DNA vector for every 50 &mu;L total volume to PCR tube 3. Add 1 &mu;L of each primer for every 50 &mu;L total volume 4. Add 5 &mu;L of dNTP for every 50 &mu;L total volume 5. Add 5 &mu;L of the correct buffer solution (depending on enzyme used) for every 50 &mu;L total volume 6a. If using PFU, use 1 &mu;L for every 50 &mu;L total volume, and the balance deionized water (add enzyme last) 6b. If using Taw, use 0.5 &mu;L for every 50 &mu;L total volume, 1.5 &mu;L 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 &mu;L total volume using Taq polymerase 35 &mu;L dd water 5 &mu;L dNTP 5 &mu;L Taq buffer 1 &mu;L Primer 1 1 &mu;L Primer 2 1 &mu;L DNA vector 1.5 &mu;L MgCl 0.5 &mu;L Taq polymerase

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

 PCR Clean Up Standard Protocol
All of our PCR clean ups are performed using QIAGEN kits and protocols.

QIAGEN's website can be found HERE.


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