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

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>Alan6017518
Line 94: Line 94:
*Add 10ul of MDA mastermix to each sample
*Add 10ul of MDA mastermix to each sample
{| {{table}}
{| {{table}}
| align="center" style="background:#f0f0f0;"|'''Plate Layout'''
| align="center" style="background:#f0f0f0;"|'''Plate Layout'''
| align="center" style="background:#f0f0f0;"|'''Plate Layout'''
| align="center" style="background:#f0f0f0;"|'''Plate Layout'''
| align="center" style="background:#f0f0f0;"|'''Plate Layout'''
|-
|-
| ||1||2
| ||1
|-
|-
| A||A1-100 Sperm Cells||C2-Noi-600pg GM12878
| A||A
|-
|-
| B||A2-100 Sperm Cells||C3-Noi-600pg GM12878
| B||B
|-
|-
| C||B1-100ESCs ||Empty
| C||C
|-
|-
| D||B2-100ESCs ||Empty
| D||D
|-
|-
| E||C1-600pg GM12878||Empty
| E||E
|-
|-
| F||D-1.2ng GM12878||Empty
| F||F
|-
|-
| G||E1-NTC||Empty
| G||Empty
|-
|-
| H||E2-NTC(MDA)||Empty
| H||Empty
|-
|-
| align="center" style="background:#f0f0f0;"|'''Plate Layout'''
| Plate Layout||Plate Layout
| align="center" style="background:#f0f0f0;"|'''Plate Layout'''
| align="center" style="background:#f0f0f0;"|'''Plate Layout'''
|}
|}


30C 60 min,  85C 3 min, 4C hold
30C 60 min,  85C 3 min, 4C hold

Revision as of 22:43, 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
  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

  • 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
  1. Step1 98C, 8m
  2. Step2 64C, 3.5hr
  3. 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 15.4
1mM N9 Primer 1 7.7
25mM dNTP 0.8 6.16
RepliPhi Phi29 (100U/ul) 1 7.7
Klenow Exo Minus (10U/ul) 0.5 3.85
H2O 4.7 36.19
Total 10 77
  • Add 10ul of MDA mastermix to each sample
Plate Layout Plate Layout
1
A A
B B
C C
D D
E E
F F
G Empty
H Empty
Plate Layout Plate Layout

30C 60 min, 85C 3 min, 4C hold