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 with Taq polymerase (but not Pfx50 polymerase), 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 (15-30 seconds is sufficient for Q5)
• Select "Run program" and select "YES" when asked about heated lid
• Wait until block has reached at least 70°C (this takes a little over 2 min)
• 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

20 μL Taq reactions

Use this for diagnostic purposes.

Ingredient	Per reaction
dH2O	14.35 μL
10X Taq buffer	2.0 μL
dNTPs	0.5 μL
Taq polymerase	0.15 μL
forward primer	1.0 μL diluted 1:10
reverse primer	1.0 μL diluted 1:10
DNA template	1.0 μL

40 μL Taq reactions

This is the standard recipe. Use it in most cases for fragment amplification and sequencing.

Ingredient	Per reaction
dH2O	28.7 μL
10X Taq buffer	4.0 μL
dNTPs	1.0 μL
Taq polymerase	0.3 μL
forward primer	2.0 μL diluted 1:10
reverse primer	2.0 μL diluted 1:10
DNA template	2.0 μL

40 μL reaction with Pfx polymerase

Use this for high-fidelity cloning.

Ingredient	Per reaction
dH2O	27 μL
10X Pfx buffer	4.0 μL
dNTPs	1.2 μL
Pfx polymerase	1.0 μL
forward primer	2.4 μL diluted 1:10
reverse primer	2.4 μL diluted 1:10
DNA template	2.0 μL

20 μL reaction with Q5 polymerase

Use this for high-fidelity cloning, especially for products >3kb.

Ingredient	Per reaction
dH2O	12.4 μL
5X Q5 buffer	4.0 μL
dNTPs	0.4 μL
Q5 polymerase	0.2 μL
forward primer	1.0 μL diluted 1:10
reverse primer	1.0 μL diluted 1:10
DNA template	1.0 μL

40 μL reaction with Q5 polymerase

Use this for high-fidelity cloning, especially for products >3kb.

Ingredient	Per reaction
dH2O	24.8 μL
5X Q5 buffer	8.0 μL
dNTPs	0.8 μL
Q5 polymerase	0.4 μL
forward primer	2.0 μL diluted 1:10
reverse primer	2.0 μL diluted 1:10
DNA template	2.0 μL

Master Mix Calculations

Ingredient	20 μL reaction with Taq polymerase	40 μL reaction with Taq polymerase	40 μL reaction with Pfx polymerase	40 μL reaction with Q5 polymerase
dH2O	16.15 μL	32.3 μL	31.3 μL	24.8 μL
10X Taq buffer	2.0 μL	4.0 μL	4.0 μL	8.0 μL
dNTPs	0.5 μL	1.0 μL	1.2 μL	0.8 μL
polymerase	0.15 μL	0.3 μL	1.0 μL	0.4 μL
forward primer	0.1 μL undiluted	0.2 μL undiluted	0.25 μL undiluted	0.2 μL undiluted
reverse primer	0.1 μL undiluted	0.2 μL undiluted	0.25 μL undiluted	0.2 μL undiluted
Total volume (without template)	19 μL	38 μL	38 μL	38 μL

Primer Dilution

• 27 μL dH₂O
• 3 μL primer

Repeat for both primers