AlanFung:LabNotes/CTCF/2011-6-30: Difference between revisions
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#E-NTC W proteinase K | #E-NTC W proteinase K | ||
#F-NTC W/O proteinase K | #F-NTC W/O proteinase K | ||
==Results== |
Revision as of 23:29, 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
- 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
Overview
- Proteinase K/Protease Digestion
- Bisulfite Conversion
- Heat Denature
- MDA
- Ethanol Precipitation
- Nextera Library Construction (Low input protocol)
- 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
- Step1 98C, 8m
- Step2 64C, 3.5hr
- 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
- 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
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
- 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 File:ZhangLab 2 2011-07-06 16hr 00min.jpg
- 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