AlanFung:LabNotes/CTCF/2011-6-30: Difference between revisions
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>Alan6017518 |
>Alan6017518 (→MDA) |
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Line 72: | Line 72: | ||
*Transfer to ice immediately | *Transfer to ice immediately | ||
==MDA== | ==MDA== | ||
*Prepare MDA Master Mix | |||
{| {{table}} | |||
| align="center" style="background:#f0f0f0;"|'''Content''' | |||
| align="center" style="background:#f0f0f0;"|'''Volume Per Reaction''' | |||
| align="center" style="background:#f0f0f0;"|'''MM''' | |||
|- | |||
| 10X RepliPhi Buffer||2||22 | |||
|- | |||
| 1mM N9 Primer||1||11 | |||
|- | |||
| 25mM dNTP||0.8||8.8 | |||
|- | |||
| RepliPhi Phi29 (100U/ul)||1||11 | |||
|- | |||
| Klenow Exo Minus (10U/ul)||0.5||5.5 | |||
|- | |||
| H2O||4.7||51.7 | |||
|- | |||
| Total||10||110 | |||
|} | |||
*Add 10ul of MDA mastermix to each sample | |||
{| {{table}} | |||
| align="center" style="background:#f0f0f0;"|'''Plate Layout''' | |||
| align="center" style="background:#f0f0f0;"|'''Plate Layout''' | |||
| align="center" style="background:#f0f0f0;"|'''Plate Layout''' | |||
|- | |||
| ||1||2 | |||
|- | |||
| A||A1-100 Sperm Cells||C2-Noi-600pg GM12878 | |||
|- | |||
| B||A2-100 Sperm Cells||C3-Noi-600pg GM12878 | |||
|- | |||
| C||B1-100ESCs ||Empty | |||
|- | |||
| D||B2-100ESCs ||Empty | |||
|- | |||
| E||C1-600pg GM12878||Empty | |||
|- | |||
| F||D-1.2ng GM12878||Empty | |||
|- | |||
| G||E1-NTC||Empty | |||
|- | |||
| H||E2-NTC(MDA)||Empty | |||
|- | |||
| align="center" style="background:#f0f0f0;"|'''Plate Layout''' | |||
| align="center" style="background:#f0f0f0;"|'''Plate Layout''' | |||
| align="center" style="background:#f0f0f0;"|'''Plate Layout''' | |||
|} | |||
30C 60 min, 85C 3 min, 4C hold |
Revision as of 22:38, 1 July 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 5min 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
MDA
- Prepare MDA Master Mix
Content | Volume Per Reaction | MM |
10X RepliPhi Buffer | 2 | 22 |
1mM N9 Primer | 1 | 11 |
25mM dNTP | 0.8 | 8.8 |
RepliPhi Phi29 (100U/ul) | 1 | 11 |
Klenow Exo Minus (10U/ul) | 0.5 | 5.5 |
H2O | 4.7 | 51.7 |
Total | 10 | 110 |
- Add 10ul of MDA mastermix to each sample
Plate Layout | Plate Layout | Plate Layout |
1 | 2 | |
A | A1-100 Sperm Cells | C2-Noi-600pg GM12878 |
B | A2-100 Sperm Cells | C3-Noi-600pg GM12878 |
C | B1-100ESCs | Empty |
D | B2-100ESCs | Empty |
E | C1-600pg GM12878 | Empty |
F | D-1.2ng GM12878 | Empty |
G | E1-NTC | Empty |
H | E2-NTC(MDA) | Empty |
Plate Layout | Plate Layout | Plate Layout |
30C 60 min, 85C 3 min, 4C hold