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

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#Filter with 5 um filter;
#Filter with 5 um filter;
#Collect the flow-through, store at 4C for the next step.
#Collect the flow-through, store at 4C for the next step.
===How to estimate cell/chr density using fluorescent microscope===
*Load cell/chr solution to a teflon coated slide, cover with a coverslip, then count the number of features per image.
*We need to estimate the volume of solution under the imaging area.
**The Hamamatsu Orca R2 camera has 1.3M pixels, and each pixel is 6.45 micron in size. Since the sensor is (almost) a square, the width should be ~1200 pixels on each sides, or 1200 x 6.45 um = 7.74 mm. Under a 20x objective, each image corresponds to a 0.39mm x 0.39mm square area.
**To estimate the height, roughly 1ul of solution can fill a well of ~5mm diameter. So the height roughly 50 um.
**Therefore, the volume is 7.6 nL. If there are 38 cells in each image, then the cell density is ~5 millions per mL.

Revision as of 06:45, 19 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.

How to estimate cell/chr density using fluorescent microscope

  • Load cell/chr solution to a teflon coated slide, cover with a coverslip, then count the number of features per image.
  • We need to estimate the volume of solution under the imaging area.
    • The Hamamatsu Orca R2 camera has 1.3M pixels, and each pixel is 6.45 micron in size. Since the sensor is (almost) a square, the width should be ~1200 pixels on each sides, or 1200 x 6.45 um = 7.74 mm. Under a 20x objective, each image corresponds to a 0.39mm x 0.39mm square area.
    • To estimate the height, roughly 1ul of solution can fill a well of ~5mm diameter. So the height roughly 50 um.
    • Therefore, the volume is 7.6 nL. If there are 38 cells in each image, then the cell density is ~5 millions per mL.