Griffitts:PCR

PCR with Taq Polymerase

Materials

• Taq polymerase (NEB)
• 10X buffer (w/Mg)
• dNTP's (10mM) (NEB)
• Template DNA
• Primers (10uM from 100uM stock)

Procedure

1. Thaw buffer, dNTP's and primers. Keep polymerase on ice throughout the procedure.
2. In standard PCR tube combine components according to the recipe below. Set the reaction up on ice.
3. Select the appropriate cycling program and verify that all of the parameters are correct.
4. Select Run program and select "YES" when asked about heated lid.
5. Wait until block has reached the beginning temperature. Place PCR tube into the machine.
6. Lower the lid until it latches and slightly tighten the heated lid onto the tubes.

Recipes

Standard reaction
37ul H2O
5ul 10X buffer
1ul dNTPs
0.4ul Taq
1ul template
3ul primer1
3ul primer2

PCR lysis buffer
5mM Tris pH8
2mM EDTA
0.5% Triton

Notes

• If performing multiple PCR's with the same primers, then just use 100uM stock, calculate for 0.3ul per reaction, and correct the volume of water.
• For amplifying rhizobium sequences, we typically boil a dense suspension of cells in PCR lysis buffer, and use this as template. Adding cells directly to the reaction may be sufficient.