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

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>Alan6017518
 
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Line 68: Line 68:
*Elute with 11ul EB Buffer
*Elute with 11ul EB Buffer
*Transfer bisulfite converted samples to 0.2ml tubes for MDA
*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==
==Heat Denature==
*92C for 30 sec
*92C for 30 sec
Line 78: Line 80:
| align="center" style="background:#f0f0f0;"|'''MM'''
| align="center" style="background:#f0f0f0;"|'''MM'''
|-
|-
| 10X RepliPhi Buffer||2||15.4
| 10X RepliPhi Buffer||2||13.2
|-
|-
| 1mM N9 Primer||1||7.7
| 1mM N9 Primer||1||6.6
|-
|-
| 25mM dNTP||0.8||6.16
| 25mM dNTP||0.8||5.28
|-
|-
| RepliPhi Phi29 (100U/ul)||1||7.7
| RepliPhi Phi29 (100U/ul)||1||6.6
|-
|-
| Klenow Exo Minus (10U/ul)||0.5||3.85
| Klenow Exo Minus (10U/ul)||0.5||3.3
|-
|-
| H2O||4.7||36.19
| H2O||4.7||31.02
|-
|-
| Total||10||77
| Total||10||66
|}
|}
*Add 10ul of MDA mastermix to each sample
*Add 10ul of MDA mastermix to each sample
Line 119: Line 121:


30C 60 min,  85C 3 min, 4C hold
30C 60 min,  85C 3 min, 4C hold
==Ethanol Precipitation ==
==Ethanol Precipitation ==
*Add 2.5X 100% Ethanol
*Add 2.5X 100% Ethanol
Line 132: Line 135:
*Add 8ul of nuclease free H2O (aliquot 2ul to perform PCR Analysis)
*Add 8ul of nuclease free H2O (aliquot 2ul to perform PCR Analysis)
*Let sit at RT to dissolve the pellet
*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
{| {{table}}
| align="center" style="background:#f0f0f0;"|'''Content'''
| align="center" style="background:#f0f0f0;"|'''Volume Per Reaction'''
| align="center" style="background:#f0f0f0;"|'''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_2011-07-06_1.jpg]]
[[File:ZhangLab_2 2011-07-06 10hr 26min.jpg|300px]]
===Results===
*All of the samples were amplified
*D had quite a bit of sample lost due to the error during bisulfite column purification
*When comparing E & F, it appears that there is a contamination problem with the proteinase K, F has less product compared to E
*When comparing A & B, it appears that using 0.1ulproteinase K yields more end product
#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
==Bisulfite efficiency PCR Analysis==
*Mix 3 MM for CHR8, 21 and 22
{| {{table}}
| align="center" style="background:#f0f0f0;"|'''Content'''
| align="center" style="background:#f0f0f0;"|'''Volume per Reaction'''
| align="center" style="background:#f0f0f0;"|'''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
#96C, 3m
#95C, 30s
#62C, 1m
#72C, 1m
#Go to step2 repeat 39 times
#72C, 5m
#4C,  Forever
===Page Analysis===
[[File:ZhangLab_2 2011-07-06 15hr 57min.jpg|400px]]
[[File:ZhangLab_2 2011-07-06 16hr 00min.jpg|400px]]
#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
===Results===
*A All failed (May be working on CHR21, cannot really tell since the image looks weird)
*B CHR22 only
*C All worked
*D CHR8 & CHR21 only
*E CHR21 only
*F CHR22 only
==To do==
*Incorporate the protease digestion after tagmentation to improve yield
*Repeat experiment without proteinase K digestion
*Repeat experiment with 100 cells with 600pg gDNA as positive control

Latest revision as of 20:42, 7 July 2011

N9-MDA Bisulfite Converted Sequencing Library Construction[edit]

Objective[edit]

  • 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[edit]

  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[edit]

Proteinase K/Protease Digestion[edit]

  • 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[edit]

  • 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[edit]

  • 92C for 30 sec
  • Transfer to ice immediately

MDA[edit]

  • 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[edit]

  • 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)[edit]

  • 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[edit]

File:ZhangLab 2011-07-06 1.jpg File:ZhangLab 2 2011-07-06 10hr 26min.jpg

Results[edit]

  • All of the samples were amplified
  • D had quite a bit of sample lost due to the error during bisulfite column purification
  • When comparing E & F, it appears that there is a contamination problem with the proteinase K, F has less product compared to E
  • When comparing A & B, it appears that using 0.1ulproteinase K yields more end product
  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[edit]

  • 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[edit]

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

Results[edit]

  • A All failed (May be working on CHR21, cannot really tell since the image looks weird)
  • B CHR22 only
  • C All worked
  • D CHR8 & CHR21 only
  • E CHR21 only
  • F CHR22 only

To do[edit]

  • Incorporate the protease digestion after tagmentation to improve yield
  • Repeat experiment without proteinase K digestion
  • Repeat experiment with 100 cells with 600pg gDNA as positive control