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.
- Remove 2 cold plates from freezer.
- 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.
- Add 12.5 ul forward primer, and 12.5 ul reverse primer to one PCR Master Mix tube, for a 96 well plate.
- 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.
- Pipette 131 ul of the mix (forward primer, reverse primer, and PCR Master Mix) into each well of the eight well strip.
- Pipette 10.5 ul of the mix into each well of a 96 well plate using a multi-channel pipette.
- 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