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.

Use this for diagnostic purposes.

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

Use this for high-fidelity cloning.

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

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

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

Repeat for both primers