Gill:S. aureus pre-treatment for plasmid preps

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Contents

Materials

  • Lysostaphin solution
  • Overnight S. aureus culture
  • Plasmid prep kit

Procedure

Lysostaphin stock preparation

1 mg/ml in 20 mM sodium acetate, pH 4.5.

  1. Make up ~50 ml of 20 mM sodium acetate in MQ water.
  2. Adjust pH to 4.5 with acetic acid or NaOH as needed.
  3. Filter sterilize solution.
  4. Dissolve whole vial of lysostaphin (1 mg) in 1 ml buffer.
  5. Aliquot solution out into 25 ul aliquots in sterile eppendorf tubes.
  6. Label and store at -20. Avoid repeated freeze-thawing of aliquots.

Cell pre-treatment for plasmid preps (Qiagen kit)

  1. Centrifuge 0.5 ml of o/n culture at 14,000 x g, 1 min.
  2. Remove supernatant. Resuspend cells in 0.25 ml of buffer P1.
  3. Add 5 ul of 1 mg/ml lysostaphin stock (20 ug/ml final).
  4. Vortex and incubate 1 h at 37 deg. C and check for clearing. If clearing is slow, try doubling lysostaphin concentration to 40 ug/ml. Suspension does not have to be completely clear.
  5. Continue with normal Qiagen kit procedure, include endA removal wash step.

Cell pre-treatment for plasmid preps (Zymo kit)

  1. Centrifuge 0.5 ml of o/n culture at 14,000 x g, 1 min.
  2. Remove supernatant. Resuspend cells in 0.6 ml TE buffer (10 mM Tris-HCl, 1 mM EDTA, pH 8).
  3. Add 6 ul of 1 mg/ml lysostaphin stock (10 ug/ml final).
  4. Vortex and incubate 1 h at 37 deg. C and check for clearing. If clearing is slow, try doubling lysostaphin concentration to 20 ug/ml. Suspension does not have to be completely clear.
  5. Continue with normal Zymo kit procedure, include endA removal wash step.

Notes

Please feel free to post comments, questions, or improvements to this protocol. Happy to have your input!

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