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