NOTE: These are the amounts that BIO uses, but I have found greater success with reducing the primer and using 7.5ul Forward Primer and 7.5ul Reverse Primer.

  1. Remove 2 cold plates from freezer.
  2. Remove the two primers that you will use in the reaction (a forward primer and a reverse primer) and PCR Master Mix tubes from freezer. Melt the liquid by holding the tubes in your hand.
  3. Add 12.5 ul forward primer, and 12.5 ul reverse primer to one PCR Master Mix tube, for a 96 well plate.
  4. Place an eight well strip onto a cold plate, and a skirted plate in the other cold plate. A plate that is “skirted” means that there is an edge around the plate.
  5. Pipette 131 ul of the mix (forward primer, reverse primer, and PCR Master Mix) into each well of the eight well strip.
  6. Pipette 10.5 ul of the mix into each well of a 96 well plate using a multi-channel pipette.
  7. Seal the plate with self-adhering plastic and store in the freezer until you are ready to use it.

PCR reaction mix 100 samples:

  • 10% trehalose 625ul
  • ddH2O 200ul
  • 10 X buffer 125ul
  • MgCl2. 62.5ul
  • dNTPs 6.25ul
  • Platinum Taq 6ul
  • Forward primer 12.5ul
  • Reverse primer 12.5ul

Total volume: 1050ul

Each reaction is 12.5ul. If you are not working on an entire plate of samples, eight well strips can be used. Each individual tube then requires:

  • PCR Master Mix 10.25ul
  • Forward Primer 0.125ul
  • Reverse Primer 0.125ul
  • Template DNA 2ul

When you have your plate made, continue to the PCR Procedure.

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Updated April 27 2009