AlanFung:LabNotes/CTCF/2011-6-30
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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
- A-600pg gDNA W 0.1ul proteinase K
- B-600pg gDNA W proteinase k
- C-600pg gDNA W/O proteinase k
- D-600pg gDNA W protease
- E-NTC W proteinase K
- F-NTC W/O proteinase K
Overview
- Proteinase K/Protease Digestion
- Bisulfite Conversion
- Heat Denature
- MDA
- Ethanol Precipitation
- Nextera Library Construction (Low input protocol)
- PAGE Analysis
Protocol
Proteinase K/Protease Digestion
- QIAGEN Protease (5AU/ml=5mAU/ul), use 0.5mAU for protein digestion
- Prepare 1:10 Protease(0.5mAU/ul)
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 |
5mAU/ul Protease | 0 | 0 | 0 | 1.2 | 0 | 0 |
UV-RT PCR Grade Water | 7.8 | 7.8 | 8.8 | 7.6 | 9 | 10 |
Total | 20 | 20 | 20 | 20 | 20 | 20 |
- Incubate the samples at 50C for 20min