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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.

40 μL

Taq

reactions

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

40 μL reaction with

Pfx

polymerase

Use this for high-fidelity cloning.

20 μL reaction with

Q5

polymerase

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


40 μL reaction with

Q5

polymerase

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

Master Mix Calculations

Primer Dilution

  • 27 μL dH2O
  • 3 μL primer

Repeat for both primers