AlanFung:LabNotes/CTCF/2011-6-30: Difference between revisions

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>Alan6017518
Line 22: Line 22:
==Protocol==
==Protocol==
==Proteinase K/Protease Digestion==
==Proteinase K/Protease Digestion==
{| {{table}}
| align="center" style="background:#f0f0f0;"|'''Content'''
| align="center" style="background:#f0f0f0;"|'''A'''
| align="center" style="background:#f0f0f0;"|'''B'''
| align="center" style="background:#f0f0f0;"|'''C'''
| align="center" style="background:#f0f0f0;"|'''D'''
| align="center" style="background:#f0f0f0;"|'''E'''
| align="center" style="background:#f0f0f0;"|'''F'''
|-
| M-Digestion Buffer (2X)||10||10||10||10||10||10
|-
| Sample||1.2||1.2||1.2||1.2||0||0
|-
| 1:10 Proteinase K||1||0||0||0||0||0
|-
| Proteinase K||0||1||0||0||1||0
|-
| Protease||0||0||0||1||0||0
|-
| UV-RT PCR Grade Water||7.8||7.8||8.8||7.8||9||10
|-
| Total||20||20||20||20||20||20
|}

Revision as of 19:04, 30 June 2011

N9-MDA Bisulfite Converted Sequencing Library Construction

Objective

  • Figure out source of contamination from 6/28 experiment
  • Repeat 6/28 experiment with only gDNA (UV EB buffer, use RT-PCR grade water as NTC)
  • Sample List
  1. A-600pg gDNA W 0.1ul proteinase K
  2. B-600pg gDNA W proteinase k
  3. C-600pg gDNA W/O proteinase k
  4. D-600pg gDNA W protease
  5. E-NTC W proteinase K
  6. F-NTC W/O proteinase K

Overview

  1. Proteinase K/Protease Digestion
  2. Bisulfite Conversion
  3. Heat Denature
  4. MDA
  5. Ethanol Precipitation
  6. Nextera Library Construction (Low input protocol)
  7. PAGE Analysis

Protocol

Proteinase K/Protease Digestion

Content A B C D E F
M-Digestion Buffer (2X) 10 10 10 10 10 10
Sample 1.2 1.2 1.2 1.2 0 0
1:10 Proteinase K 1 0 0 0 0 0
Proteinase K 0 1 0 0 1 0
Protease 0 0 0 1 0 0
UV-RT PCR Grade Water 7.8 7.8 8.8 7.8 9 10
Total 20 20 20 20 20 20