Griffitts:PCR

PCR with Taq Polymerase

Procedure
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.
 * 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

25 μL reactions
Use this for diagnostic purposes.

40 μL reactions
This is the standard recipe. Use it in most cases for fragment amplification.

40 μL reaction with Pfx polymerase
Use this for high-fidelity cloning.

Primer Dilution
Repeat for both primers
 * 27 μL dH2O
 * 3 μL primer