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  • Protein expression using the K. lactis Protein Expression Kit - Cloning a PCR fragment into pKLAC2.

    Introduction

    The procedure below is for cloning a fragment produced by PCR into pKLAC2.  This example assumes that the PCR fragment contains a 5' XhoI site, and has a stop codon, followed by a NotI site incorporated into its 3' end.

    Protocol

    1. Prepare a PCR fragment of the gene of interest.
    2. Digest 0.5 μg of pKLAC2 DNA with 10 units of XhoI and 10 units of NotI in 20 μl of 1X NEBuffer 3 (supplied at 10X stock) supplemented with 100 μg/ml BSA (supplied as a 100 mg/ml stock) at 37oC for 2 hours.
    3. Digest 0.5 μg of the PCR fragment with 10 units of XhoI and 10 units of NotI in 20 μl of 1X NEBuffer 3 supplemented with 100 μg/ml BSA at 37oC for 2 hours.
    4. Add an equal volume of phenol:chloroform (1:1, v/v) to the restriction digests, mix and remove the aqueous (top) phase to a fresh tube.  Repeat using only chloroform.

      Alternatively, the DNA fragments can be isolated using one of the many commercially available fragmentation purification kits.  If a kit is used, skip to step 8.
    5. Add 10 μg glycogen or tRNA as carrier to both digests, then add a 1/10th volume 3M sodium acetate, mix and add an equal volume of 100% isopropanol.  Incubate at room temperature for 10 minutes.
    6. Microcentrifuge at 12,000 x g for 15 minutes.  Pour off the supernatant and gently rinse the pellet with 70% ethanol.  Allow the pellet to air-dry (~10 minutes).
    7. Resuspend each sample in 25 μl of 10 mM Tris-HCl, 1 mM EDTA (pH 8.0).
    8. Mix

      • 2 μl pKLAC2 digest (~40 ng)
      • 10 μl PCR fragment (insert digest) (~200 ng)
      • 2 μl 10X T4 DNA Ligase Buffer
      • 14 μ deionized water
      • 1 μl (~400 units) T4 DNA Ligase (NEB #M0202)
    9. Incubate at 16oC for 2 hours to overnight.

      Completed ligation reactions can be stored frozen at -20oC indefinitely prior to transformation.
    10. Transformation of frozen competent NEB 5-aplha F' lq Competent E. coli (NEB #C2992) is recommended using 2-4 μl of the ligation reaction.

      Any competent E. coli strain can be used.  Blue-white screening, however, is not possible with pKLAC2.
    11. Prepare miniprep DNA from several transformants.  Digest each vector with an appropriate restriction endonuclease to determine the presence of a cloned insert.

      Expression vectors may be stored at -20oC indefinitely.