AlanFung:LabNotes/CTCF/2011-6-28: Difference between revisions
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| align="center" style="background:#f0f0f0;"|'''Plate Layout''' | | align="center" style="background:#f0f0f0;"|'''Plate Layout''' | ||
|} | |} | ||
Content Volume Per Reaction MM | {| {{table}} | ||
10X RepliPhi Buffer 2 22 | | align="center" style="background:#f0f0f0;"|'''Content''' | ||
1mM N9 Primer 1 11 | | align="center" style="background:#f0f0f0;"|'''Volume Per Reaction''' | ||
25mM dNTP 0.8 8.8 | | align="center" style="background:#f0f0f0;"|'''MM''' | ||
RepliPhi Phi29 (100U/ul) 1 11 | |- | ||
Klenow Exo Minus (10U/ul) 0.5 5.5 | | 10X RepliPhi Buffer||2||22 | ||
H2O 4.7 51.7 | |- | ||
Total 10 110 | | 1mM N9 Primer||1||11 | ||
|- | |||
| 25mM dNTP||0.8||8.8 | |||
|- | |||
| RepliPhi Phi29 (100U/ul)||1||11 | |||
|- | |||
| Klenow Exo Minus (10U/ul)||0.5||5.5 | |||
|- | |||
| H2O||4.7||51.7 | |||
|- | |||
| Total||10||110 | |||
|} | |||
30C 60 min, 85C 3 min, 4C hold | 30C 60 min, 85C 3 min, 4C hold | ||
Revision as of 00:26, 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 |
Content | Volume Per Reaction | MM |
10X RepliPhi Buffer | 2 | 22 |
1mM N9 Primer | 1 | 11 |
25mM dNTP | 0.8 | 8.8 |
RepliPhi Phi29 (100U/ul) | 1 | 11 |
Klenow Exo Minus (10U/ul) | 0.5 | 5.5 |
H2O | 4.7 | 51.7 |
Total | 10 | 110 |
30C 60 min, 85C 3 min, 4C hold