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

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#4C,  Forever
#4C,  Forever
==Page Analysis==
==Page Analysis==
[[File:ZhangLab_2 2011-07-06 15hr 57min.jpg|300px]]
[[File:ZhangLab_2 2011-07-06 15hr 57min.jpg|400px]]
[[File:ZhangLab_2 2011-07-06 16hr 00min.jpg|300px]]
[[File:ZhangLab_2 2011-07-06 16hr 00min.jpg|400px]]

Revision as of 23:23, 6 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
  • Tube D has higher volume after elution, probably due to extra wash buffer loaded into tube

Heat Denature

  • 92C for 30 sec
  • Transfer to ice immediately

MDA

  • Prepare MDA Master Mix
Content Volume Per Reaction MM
10X RepliPhi Buffer 2 13.2
1mM N9 Primer 1 6.6
25mM dNTP 0.8 5.28
RepliPhi Phi29 (100U/ul) 1 6.6
Klenow Exo Minus (10U/ul) 0.5 3.3
H2O 4.7 31.02
Total 10 66
  • 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

Ethanol Precipitation

  • Add 2.5X 100% Ethanol
  • Add 0.1X 3M NaOAC
  • Add 1ul glycoblue
  • Store at -80C for 30min (CONTINUE ON 7/5)
  • Spin down at 10,000rpm at 4C for 15min
  • Remove all liquid
  • Add 500ul 4C chilled 75% ethanol
  • Spin at 10,000rpm at 4C for 5 min
  • Remove all liquid
  • Let dry in hood for 5 min
  • Add 8ul of nuclease free H2O (aliquot 2ul to perform PCR Analysis)
  • Let sit at RT to dissolve the pellet
  • Sucked pellet through the vacuum, only a small piece left

Nextera Library Construction (Low input protocol)

  • In a qPCR tube, mix
  • 3ul genomic DNA
  • 1ul 1:5 Nextera illumina compatible transposomes (10 μl Nextera enzyme, 20 μl 1XTE, 20 μl 100% glycerol)
  • 1ul 5X NExtera HMW buffer
  • Incubate at 55C for 5 min
  • Setup PCR Reaction with Klenow Exo- but without primers
Content Volume Per Reaction MM
Water 17.5 115.5
DNA 5 Do not add to MM
KAPA Supermix 25 165
Klenow Exo Minus 0.5 3.3
10uM Orange Primer 1 Do not add to MM
10uM Blue Primer 1 Do not add to MM
Total 50 283.8
  • Add 43ul MM to each reaction
  • Incubate reactions at RT for 5 min
  • Put the tubes back on ice
  • Add Primers
  • Perform thermal cycling
Perform Kapa PCR Reaction
95C 3m 
95C 10s
58C 30s
72C 3m
plate read
Goto step 2 repeat 34
72C 10m
Hold 15C

PAGE Analysis

File:ZhangLab 2 2011-07-06 10hr 26min.jpg

  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

Bisulfite efficiency PCR Analysis

  • Mix 3 MM for CHR8, 21 and 22
Content Volume per Reaction MM
2X iQ Super Mix 20 132
3.3uM Primer F 6 39.6
3.3uM Primer R 6 39.6
gDNA 2 Do not add to MM
H2O 6 39.6
Total 40 264

Perform PCR reaction in thermocycler

  1. 96C, 3m
  2. 95C, 30s
  3. 62C, 1m
  4. 72C, 1m
  5. Go to step2 repeat 39 times
  6. 72C, 5m
  7. 4C, Forever

Page Analysis

File:ZhangLab 2 2011-07-06 15hr 57min.jpg File:ZhangLab 2 2011-07-06 16hr 00min.jpg