Kun:LabNotes/Haplotyping/2010-8-18: Difference between revisions

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#Will prepare chromosomes for in-well amplification by Jeff.
#Will prepare chromosomes for in-well amplification by Jeff.
#Will also perform in-tube amplification by myself.
#Will also perform in-tube amplification by myself.
===Chromosome prep===
#Make the chromosome isolation buffer (CIB):
    100mM MgSO4  1.0ml
    KCl/HEPES    9.0ml
    RNase        0.5ml
    120mM DTT    0.25ml
#Collected metaphase blocked cells from 1/2 of a T25 flask (~8ml);
#Spin at 800 rpm for 8 min at 4C.
#Remove all supernatant;
#Flick bottom of tube several times to lossen pellet;
#Add 1ml CIM, flick several times to mix, then incubate at RT for 10 min;
#Add 0.1ml 2.5% Triton X-100;
#Incubate at RT for 10 min;
#Syringe 5x with a 22 guage needle;
#Incubate at 37C for 30min;
#Filter with 5 um filter;
#Collect the flow-through, store at 4C for the next step.

Revision as of 17:27, 18 August 2010

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.

Chromosome prep

  1. Make the chromosome isolation buffer (CIB):
    100mM MgSO4   1.0ml
    KCl/HEPES     9.0ml
    RNase         0.5ml
    120mM DTT     0.25ml
  1. Collected metaphase blocked cells from 1/2 of a T25 flask (~8ml);
  2. Spin at 800 rpm for 8 min at 4C.
  3. Remove all supernatant;
  4. Flick bottom of tube several times to lossen pellet;
  5. Add 1ml CIM, flick several times to mix, then incubate at RT for 10 min;
  6. Add 0.1ml 2.5% Triton X-100;
  7. Incubate at RT for 10 min;
  8. Syringe 5x with a 22 guage needle;
  9. Incubate at 37C for 30min;
  10. Filter with 5 um filter;
  11. Collect the flow-through, store at 4C for the next step.