Griffitts:PCR

PCR with Taq Polymerase

Procedure

• Prepare your template sample
  • For amplifying Sinorhizobium sequences, boil a dense suspension of cells in 35 μL PCR lysis buffer for ~2 min and vortex
  • For amplifying E. coli, adding cells directly to the reaction is sufficient
• Thaw the Taq buffer, dNTPs, and primers
  • Keep Taq polymerase on ice throughout the procedure
• Combine components according to one of the recipes below
  • Set the reaction up on ice
• Select the appropriate cycling program and verify that all of the parameters are correct
  • Allow 1 minute of extension time per 1 KB DNA being amplified
• 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

NOTE: When the PCR machine has finished cycling, your samples may be stored at -20°C. You may now proceed to gel electrophoresis and/or PCR clean-up.

Reaction recipes

25 μL reactions

Use this for diagnostic purposes.

Ingredient	Per reaction
dH2O	17.8 μL
10X Taq buffer	2.5 μL
dNTPs	0.5 μL
Taq polymerase	0.2 μL
DNA template	1 μL
forward primer	1.5 μL diluted 1:10
reverse primer	1.5 μL diluted 1:10

40 μL reactions

This is the standard recipe. Use it in most cases for cloning.

Ingredient	Per reaction
dH2O	29.1 μL
10X Taq buffer	4 μL
dNTPs	0.8 μL
Taq polymerase	0.3 μL
DNA template	1 μL
forward primer	2.4 μL diluted 1:10
reverse primer	2.4 μL diluted 1:10

40 μL reaction with Pfx polymerase

Use this for high-fidelity cloning.

Ingredient	Per reaction
dH2O	27.4 μL
10X Pfx buffer	4.0 μL
dNTPs	1.2 μL
Pfx polymerase	1.0 μL
DNA template	1.6 μL
forward primer	2.4 μL diluted 1:10
reverse primer	2.4 μL diluted 1:10

Master Mix Calculations

Ingredient	25 μL reaction with Taq polymerase	40 μL reaction with Taq polymerase	40 μL reaction with Pfx polymerase
dH2O	20.5 μL	33.4 μL	32.3
10X Taq buffer	2.5 μL	4.0 μL	4.0 μL
dNTPs	0.5 μL	0.8 μL	1.2 μL
polymerase	0.2 μL	0.3 μL	1.0 μL
forward primer	0.15 μL undiluted	0.24 μL undiluted	0.24 μL undiluted
reverse primer	0.15 μL undiluted	0.24 μL undiluted	0.24 μL undiluted
Total volume (without DNA)	24 μL	39 μL	38.4 μL

Primer Dilution

• 27 μL dH₂O
• 3 μL primer

Repeat for both primers