AlanFung:LabNotes/CTCF/2011-6-28: Difference between revisions

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| align="center" style="background:#f0f0f0;"|'''Plate Layout'''
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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
30C 60 min,  85C 3 min, 4C hold



Revision as of 00:25, 28 June 2011

MDA Bisulfite Converted Sequencing Library Consturction

Objective

  • Work on sperm/ESCs control cell samples plus GM12878 gDNA
  • Sample List
  1. A1-100 sperm cells
  2. A2-100 sperm cells (repeat)
  3. B1-100 ESCs
  4. B2-100 ESCs (repeat)
  5. C-600pg GM12878 gDNA
  6. D-1.2ng GM12878 gDNA
  7. 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
  1. Sperms: ~50cell/tube; 15 tubes
  2. 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

Ethanol Precipitation

Nextera Library Construction (Low input protocol)

PAGE Analysis

Sanger Analysis