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, or 0.19mm x 0.19mm under a 40x objective.
**To estimate the height, roughly 5ul of solution can fill a well of ~12mm diameter. So the height is 5ul/(3.14x6mm^2)= 44 um. 
                  20x objective    40x objective
      Area      0.39mm x 0.39mm  0.19mm x 0.19mm
      Volume        6.62uL            1.66nL
===Examination of chromosome prep===
*After disrupt the cells with syringe (i did it 20 times), the solution was filtered with 5 a micron filter.
*I then spin the solution at 5000g for 5 minutes. A small white pallet is visible at the bottom of the tube. I transfer 800ul of the supernatant to another tube (Sup1).
*I resuspended the pallet with the remaining ~200ul supernatant, repeat the centrifugation, transfer all supernatant to another tube (Sup2), and resuspended the pallet with 100ul of CIB.
*I stained the chromosome suspension as well as Sup1/Sup2 with SYBG I, and checked the morphology with IX81.
**The chromosome suspension contains small highly compact chromosomes, plus some larger objects, which could be nucleus or aggregates of chromosomes. There are ~35 chromosomes in each image taken with 40x objective, which is equivalent to 21 millions chromosomes per mL.  Since I made an 1:2 dilution for staining/imaging, the actual density is ~42M/ml.
**Interestingly, Sup1 is not blank. There are as many less compact chromosomes (~35 per image). So the density is also ~21M/mL.
              Chromosome suspension                            Sup1
  [[Image:2010-08-19-chr-prep-40x-2.png|350px]][[Image:2010-08-19-Sup1-40x-1.png|350px]]

Latest revision as of 19:13, 19 August 2010

Human chromosome preparation[edit]

Make buffers[edit]

  • 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[edit]

  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[edit]

  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[edit]

  • 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, or 0.19mm x 0.19mm under a 40x objective.
    • To estimate the height, roughly 5ul of solution can fill a well of ~12mm diameter. So the height is 5ul/(3.14x6mm^2)= 44 um.
                 20x objective     40x objective
     Area       0.39mm x 0.39mm   0.19mm x 0.19mm
     Volume         6.62uL            1.66nL

Examination of chromosome prep[edit]

  • After disrupt the cells with syringe (i did it 20 times), the solution was filtered with 5 a micron filter.
  • I then spin the solution at 5000g for 5 minutes. A small white pallet is visible at the bottom of the tube. I transfer 800ul of the supernatant to another tube (Sup1).
  • I resuspended the pallet with the remaining ~200ul supernatant, repeat the centrifugation, transfer all supernatant to another tube (Sup2), and resuspended the pallet with 100ul of CIB.
  • I stained the chromosome suspension as well as Sup1/Sup2 with SYBG I, and checked the morphology with IX81.
    • The chromosome suspension contains small highly compact chromosomes, plus some larger objects, which could be nucleus or aggregates of chromosomes. There are ~35 chromosomes in each image taken with 40x objective, which is equivalent to 21 millions chromosomes per mL. Since I made an 1:2 dilution for staining/imaging, the actual density is ~42M/ml.
    • Interestingly, Sup1 is not blank. There are as many less compact chromosomes (~35 per image). So the density is also ~21M/mL.
             Chromosome suspension                             Sup1
  File:2010-08-19-chr-prep-40x-2.pngFile:2010-08-19-Sup1-40x-1.png