AlanFung:LabNotes/CTCF/2011-6-28: Difference between revisions
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>Alan6017518 (→MDA) |
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*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
- 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
- Sperms: ~50cell/tube; 15 tubes
- 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