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Overlap Extension

Introduction

This procedure is used to generate a construct for making an in-frame deletion of a gene. It involves first amplifying DNA at either end of the target gene. The inside primers are designed to overlap each other so that in the second round of PCR the two can be zipped together. The resulting clone will then be ligated into pJQ200SK for deletion of the target gene.

Round One

  • 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 Pfx50 buffer, dNTPs, and primers
    • Keep Pfx50 polymerase on ice throughout the procedure
  • Combine components according to  the First Round recipe below
    • Set the reaction up on ice
    • You will need to set up separate reactions for amplifying the upstream fragment and the downstream fragment
  • 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 then place PCR tubes 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 .

Round One recipe

Round One cycling

Round Two

  • Thaw the Pfx50 buffer, dNTPs, and primers
    • Keep Pfx50 polymerase on ice throughout the procedure
  • Combine components according to  the Second Round recipe below
  • 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 then place PCR tubes 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 .

Round Two recipe

Round Two cycling

Primer Dilution

  • 27 μL dH2O
  • 3 μL primer

Repeat for each primer