AlanFung:LabNotes/CTCF/2011-6-28: Difference between revisions
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==MDA== | ==MDA== | ||
{| {{table}} | |||
| align="center" style="background:#f0f0f0;"|'''Plate Layout''' | |||
| align="center" style="background:#f0f0f0;"|'''Plate Layout''' | |||
| align="center" style="background:#f0f0f0;"|'''Plate Layout''' | |||
|- | |||
| ||1||2 | |||
|- | |||
| A||A1-100 Sperm Cells||Noi-600pg GM12878 | |||
|- | |||
| B||A2-100 Sperm Cells||Noi-600pg GM12878 | |||
|- | |||
| C||B1-100ESCs ||Empty | |||
|- | |||
| D||B2-100ESCs ||Empty | |||
|- | |||
| E||C-600pg GM12878||Empty | |||
|- | |||
| F||D-1.2ng GM12878||Empty | |||
|- | |||
| G||E1-NTC||Empty | |||
|- | |||
| H||E2-NTC(MDA)||Empty | |||
|- | |||
| Plate Layout||Plate Layout||Plate Layout | |||
|} | |||
30C 60 min, 85C 3 min, 4C hold | 30C 60 min, 85C 3 min, 4C hold | ||
Revision as of 00:20, 28 June 2011
MDA Bisulfite Converted Sequencing Library Consturction
Objective
- Work on sperm/ESCs control cell samples plus GM12878 gDNA
- Sample List
- A1-100 sperm cells
- A2-100 sperm cells (repeat)
- B1-100 ESCs
- B2-100 ESCs (repeat)
- C-600pg GM12878 gDNA
- D-1.2ng GM12878 gDNA
- E-NTC
Overview
- Proteinase K Digestion
- Bisulfite conversion
- MDA
- EtOH precipitation
- Nextera Library Construction (Low input protocol)
- Page Analysis
- Sanger Sequencing
Protocol
Proteinase K Digestion
- Label tubes A1, A2, B1, B2
- Sperms: ~50cell/tube; 15 tubes
- ES cells: ~50cell/tube; 15 tubes
- Thaw 4 tubes on ice, we will use 100cells per reaction
Bisulfite Conversion
- Setup two more 600pg GM12878 gDNA reactions for Noi to perform capture using the 330k probe set
- Proceed with Zymo column based method with improvement tweak protocol
- Elute with 11ul EB Buffer
Heat Denature
- 92C for 30 sec
- Transfer to ice immediately
MDA
Plate Layout | Plate Layout | Plate Layout |
1 | 2 | |
A | A1-100 Sperm Cells | Noi-600pg GM12878 |
B | A2-100 Sperm Cells | Noi-600pg GM12878 |
C | B1-100ESCs | Empty |
D | B2-100ESCs | Empty |
E | C-600pg GM12878 | Empty |
F | D-1.2ng GM12878 | Empty |
G | E1-NTC | Empty |
H | E2-NTC(MDA) | Empty |
Plate Layout | Plate Layout | Plate Layout |
30C 60 min, 85C 3 min, 4C hold