Griffitts:PCR

PCR with Taq Polymerase

Materials

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

Procedure

• Prepare your template sample
  • For amplifying rhizobium sequences, boil a dense suspension of cells in PCR lysis buffer.
  • For amplifying E. coli, adding cells directly to the reaction is sufficient.
• Thaw buffer, dNTPs and primers. Keep polymerase on ice throughout the procedure.
• In standard PCR tube combine components according to the recipe below. Set the reaction up on ice.
  • When running multiple samples, a Master Mix, which includes everything except the except the template and the primers (add 43.4 μL Master Mix to each tube)
  • 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.
• Select the appropriate cycling program and verify that all of the parameters are correct.
• Select "Run program" and select "YES" when asked about heated lid.
• Wait until block has reached the beginning temperature. Place PCR tube into the machine.
• Lower the lid until it latches and slightly tighten the heated lid onto the tubes.

Standard reaction recipe

37 μl H₂O
5 μl 10X Taq buffer
1 μl dNTPs
0.4 μl Taq polymerase
1 μl template
3 μl forward primer
3 μl reverse primer