Kun:LabNotes/Haplotyping/Protocols

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Lysing mammalian cells[edit]

  • Reagents:

Lysis buffer 0.45% Tween, 0.45% NP40, 2.5mM MgCl2, 50mM KCl, 10mM Tris-Cl pH8.3, 100ug/ml Proteinase K

  Tween20			225ul
  NP40				225ul
  1M MgCl2			125ul
  1M KCl			2.5ml
  1M Tris-Cl pH8.3		0.5ml
  20ug/ul Prot K		250ul*
  dH2O				46.2ml
  * Add Proteinase K just before use, 5ul stock per ml of lysis buffer used.
  • Protocol:
  1. Wash cells with cold PBS.
  2. Resuspend at 10000 cells/ul in lysis buffer.
  3. Incubate at 56 C for 1 hour.
  4. Boil for 10 minutes.
  5. Store lysate at -20 C
  • Another lysis buffer recipe
   20   mM EDTA
   10   mM Tris 8.0
   200  mM NaCl
   0.2% Triton X-100
   100  mg/ml PK
   Incubate at 37C for 1.5 hour.