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Whole-Cell PCR Protocol

Introduction

To facilitate screening of large numbers of transformants, set up PCR reactions using freshly grown cells as a source of chromosomal DNA template.

Protocol

  1. Transform K. lactis GG799 Competent Cells with a SacII (or BstXI) linearized pKLAC1-based expression construct.
  2. Streak or patch 10-20 colonies onto fresh YCB Agar Medium plates containing 5 mM acetamide (1.0 cm2 patches are recommended).
  3. Incubate at 30°C for 1-2 days.
  4. Immediately transfer the thawed cell stock to the flask containing the equilibrated growth media.
  5. Use 15 µl of lyticase-treated cells as template in a 100 µl PCR reaction containing either Integration Primers 1 and 2 (to detect proper pKLAC1 integration) or Integration Primers 2 and 3 (to detect multiple integration). Mix all reaction tubes containing cells by vortexing. The following conditions are recommended for the PCR reaction. Note that the Taq DNA Polymerase (NEB #M0267 or NEB #M0273) is added during the 80°C incubation.
  6. Analyze 10 µl of each amplification reaction on a 1% agarose gel.
  7. Test strains harboring a properly integrated expression fragment for secretion of the protein of interest.