Kun:LabNotes/Haplotyping/2010-8-18: Difference between revisions
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m (Created page with '==Human chromosome preparation== ===Make buffers=== *55mM KCl + 5.5mM HEPES @ pH8.0 *RNase: 3mg/ml *DDT: 120mM *100mM MgSO4 *2.5% Triton X-100 *Cell lysis buffer: 20mM EDTA, 10mM…') |
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*55mM KCl + 5.5mM HEPES @ pH8.0 | *55mM KCl + 5.5mM HEPES @ pH8.0 | ||
*RNase: 3mg/ml | *RNase: 3mg/ml | ||
* | *DTT: 120mM | ||
*100mM MgSO4 | *100mM MgSO4 | ||
*2.5% Triton X-100 | *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 | *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. |
Revision as of 16:52, 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
- 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.