Griffitts:PCR

PCR with Taq Polymerase

Materials

• Taq polymerase (NEB)
• 10X buffer (w/Mg²⁺)
• dNTPs (10 mM) (NEB)
• Template DNA
• Primers (10 μM from 100 μM stock)

Procedure

1. Prepare
2. Thaw buffer, dNTPs and primers. Keep polymerase on ice throughout the procedure.
3. In standard PCR tube combine components according to the recipe below. Set the reaction up on ice.
   1. When running multiple samples, a Master Mix, which includes everything except the except the template and the primers
   2. Add 43.4 μL Master Mix to each tube
4. Select the appropriate cycling program and verify that all of the parameters are correct.
5. Select Run program and select "YES" when asked about heated lid.
6. Wait until block has reached the beginning temperature. Place PCR tube into the machine.
7. Lower the lid until it latches and slightly tighten the heated lid onto the tubes.

Recipes

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

PCR lysis buffer
5 mM Tris pH 8.0
2 mM EDTA
0.5% Triton
10 mM dNTP solution

• Thaw and mix 100 mM stock solutions of G, A, T, C (NEB)
• Combine in a 1.5-mL tube:
  • 60 μl H₂O
  • 10 μl G
  • 10 μl A
  • 10 μl T
  • 10 μl C

Notes

• If performing multiple PCRs with the same primers, then just use 100 μM stock, calculate for 0.3 μl 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.