Kun:LabNotes/Haplotyping/2010-8-18

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Human chromosome preparation

Make buffers

  • 55mM KCl + 5.5mM HEPES @ pH8.0
  • RNase: 3mg/ml
  • DTT: 120mM
  • 100mM MgSO4
  • 2.5% Triton X-100
  • Cell lysis buffer: 20mM EDTA, 10mM Tris.HCl (pH 8.0), 200mM NaCl, 0.2% Triton X-100, 100mg/ml Protease

Plan

  1. Prepare metaphase chromosomes from GM20431.
  2. Also harvest unblocked cells, count them, and lyse the cells to prepare HMW gDNA.
  3. Chromosomes will be digested with Qiagen Protease prior to MDA.
  4. Digestion of chromosomes will be verified by visual inspection of the morphology under fluorescent microscope with DAPI/YOYO-1 staining.
  5. Will prepare chromosomes for in-well amplification by Jeff.
  6. Will also perform in-tube amplification by myself.