AlanFung:LabNotes/CTCF/2011-6-30: Difference between revisions
Jump to navigation
Jump to search
>Alan6017518 |
>Alan6017518 |
||
Line 22: | Line 22: | ||
==Protocol== | ==Protocol== | ||
==Proteinase K/Protease Digestion== | ==Proteinase K/Protease Digestion== | ||
*proteinase k | |||
*cells | |||
*200 5million cells 20ul proteinase k 20mg/ml 400mg | |||
*Protease | |||
20mg/ul | |||
56C | |||
*QIAGEN Protease (5AU/ml=5mAU/ul), use 0.5mAU for protein digestion | *QIAGEN Protease (5AU/ml=5mAU/ul), use 0.5mAU for protein digestion | ||
*Prepare 1:10 Protease(0.5mAU/ul) | *Prepare 1:10 Protease(0.5mAU/ul) | ||
Line 47: | Line 56: | ||
| Total||20||20||20||20||20||20 | | Total||20||20||20||20||20||20 | ||
|} | |} | ||
*Incubate | *Incubate all samples at 50C for 20min | ||
*incubate tube D at | *incubate tube D at 70C for 15min for inactivation | ||
==Bisulfite Conversion== | ==Bisulfite Conversion== | ||
*Add 130ul CT Conversion Reagent to all samples | *Add 130ul CT Conversion Reagent to all samples |
Revision as of 21:47, 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
- 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
- proteinase k
- cells
- 200 5million cells 20ul proteinase k 20mg/ml 400mg
- Protease
20mg/ul 56C
- 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 |
0.5mAU/ul 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 |
- Incubate all samples at 50C for 20min
- incubate tube D at 70C for 15min for inactivation
Bisulfite Conversion
- Add 130ul CT Conversion Reagent to all samples
- Perform reaction in thermocycler
- Step1 98C, 8m
- Step2 64C, 3.5hr
- Step3 4C, storage for up to 20 hr
- Proceed with Zymo column based method with improvement tweak protocol
- Elute with 11ul EB Buffer
- Transfer bisulfite converted samples to 0.2ml tubes for MDA
Heat Denature
- 92C for 30 sec
- Transfer to ice immediately