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

From ZhangLabWiki
Jump to navigation Jump to search
>Alan6017518
>Alan6017518
Line 27: Line 27:
*Proceed with Zymo column based method with improvement tweak protocol
*Proceed with Zymo column based method with improvement tweak protocol
*Elute with 11ul EB Buffer
*Elute with 11ul EB Buffer
==Heat Denature==
*92C for 30 sec
*Transfer to ice immediately


==MDA==
==MDA==

Revision as of 00:08, 28 June 2011

MDA Bisulfite Converted Sequencing Library Consturction

Objective

  • Work on sperm/ESCs control cell samples plus GM12878 gDNA
  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

  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

  • 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

30C 60 min, 85C 3 min, 4C hold

Ethanol Precipitation

Nextera Library Construction (Low input protocol)

PAGE Analysis

Sanger Analysis