AlanFung:LabNotes/CTCF/2011-6-30

From ZhangLabWiki
Revision as of 22:41, 1 July 2011 by >Alan6017518 (→‎MDA)
Jump to navigation Jump to search

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 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