Protein Expression using the K. lactis Protein Expression Kit - Identification of properly integrated cells

Overview

Transformants in which the expression cassette has correctly integrated into the K. lactis genome can be identified by PCR using supplied Integration Primers 1 and 2 to amplify a 2.4 kb product.  To facilitate simultaneous screening of many transformants, PCR using freshly grown cells as a course of template chromosomal DNA is recommended.

Protocol

  1. For each transformant patched and grown on YCB Agar Medium plates containing 5 mM acetamide, harvest cells from an area approximately 1 mm2 by scraping with a pipette tip and resuspend the cells in 25 μl of 1 M sorbitol containing 2 mg/ml Lyticase (Sigma #L-2524).  Mix by vortexing.  Incubate at 30oC for 1 hour.
  2. Lyse the Lyticase-treated cells in a thermocycler at 98oC for 10 minutes.
  3. Mix
    25 μl - Lyticase treated cells
    10 μl - 10X Integration Primer 1
    10 μl - 10X Integration Primer 2
    10 μl - 2mM dNTPs (NEB #N0447 , 10 mM stock)
    10 μl - 10X ThermPol Buffer (NEB #B9004 )
    1 μl   - Taq DNA Polymerase (NEB #M0267 or NEB #M0273 )
    34 μl - deionized water
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    100 μl - final reaction volume
  4. Thermocycling should consist of 30 rounds (94oC for 30 seconds, 50oC for 30 seconds and 72oC for 2 minutes), followed by incubation at 72oC for 10 minutes.
  5. Analyze 10 μl of each amplification on a 1% agarose gel.

    Integration of the expression fragment at the LAC4 locus in the K. lactis genome will result in amplification of a 2.4 kb product.