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

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#F-NTC W/O proteinase K
#F-NTC W/O proteinase K
==Bisulfite efficiency PCR Analysis==
==Bisulfite efficiency PCR Analysis==
*Mix 3 MM for CHR8, 21 and 22
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Revision as of 18:21, 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