AlanFung:LabNotes/CTCF/2011-6-28: Difference between revisions
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==To do== | ==To do== | ||
*Size Selection on samples A1-E1 (use clone well gel to extract small amount of sample to do cloning) | *Size Selection on samples A1-E1 (use clone well gel to extract small amount of sample to do cloning) | ||
*Do cloning on | *Do cloning on size selected A1-E1 samples | ||
*Repeat experiment with only gDNA (UV EB buffer, use RT-PCR grade water as NTC) | *Repeat experiment with only gDNA (UV EB buffer, use RT-PCR grade water as NTC) | ||
# gDNA without proteinase k | # gDNA without proteinase k |
Revision as of 23:47, 29 June 2011
N9-MDA Bisulfite Converted Sequencing Library Consturction
Objective
- Work on sperm/ESCs control cell samples plus GM12878 gDNA
- Sample List
- A1-100 sperm cells
- A2-100 sperm cells (repeat)
- B1-100 ESCs
- B2-100 ESCs (repeat)
- C1-600pg GM12878 gDNA
- C2-600pg GM12878 gDNA>Noi
- C3-600pg GM12878 gDNA>Noi
- D-1.2ng GM12878 gDNA
- E-NTC
Overview
- Proteinase K Digestion
- Bisulfite conversion
- MDA
- EtOH precipitation
- Nextera Library Construction (Low input protocol)
- Page Analysis
- Sanger Sequencing
Protocol
Proteinase K Digestion
- Preparation of Proteinase K
- Add 260 µl (D5020) of Proteinase K Storage Buffer to the tube containing Proteinase K. Dissolve completely
- Label tubes A1, A2, B1, B2
- Sperms: ~50cell/tube; 15 tubes
- ES cells: ~50cell/tube; 15 tubes
- Thaw 4 tubes on ice, we will use 100cells per reaction
- Spin down tubes and add 9ul nuclease free water and transfer cells to 0.2ml tubes
Content | Volume per Reaction |
M-Digestion Buffer (2X) | 10 |
Sample | 9 |
Proteinase K | 1 |
Total | 20 |
- Incubate the samples at 50C for 20min
Bisulfite Conversion
- Dilute GM12878 gDNA from 50ng/ul to 0.5ng/ul (1:100)
- Setup two more 600pg GM12878 gDNA reactions for Noi to perform capture using the 330k probe set
0.5ng/ul GM12878 gDNA | Volume Per Reaction | Make up to 20ul with H2O |
600pg | 1.2 | 18.8 |
1.2ng | 2.4 | 17.6 |
- 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
Heat Denature
- 92C for 30 sec
- Transfer to ice immediately
MDA
- Prepare MDA Master Mix
Content | Volume Per Reaction | MM |
10X RepliPhi Buffer | 2 | 22 |
1mM N9 Primer | 1 | 11 |
25mM dNTP | 0.8 | 8.8 |
RepliPhi Phi29 (100U/ul) | 1 | 11 |
Klenow Exo Minus (10U/ul) | 0.5 | 5.5 |
H2O | 4.7 | 51.7 |
Total | 10 | 110 |
- Add 10ul of MDA mastermix to each sample
Plate Layout | Plate Layout | Plate Layout |
1 | 2 | |
A | A1-100 Sperm Cells | Noi-600pg GM12878 |
B | A2-100 Sperm Cells | Noi-600pg GM12878 |
C | B1-100ESCs | Empty |
D | B2-100ESCs | Empty |
E | C-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
Ethanol Precipitation
- Add 2.5X 100% Ethanol
- Add 0.1X 3M NaOAC
- Add 1ul glycoblue
- Store at -80C for 30min (CONTINUE ON 6/29)
- 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
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 | 154 |
DNA | 5 | Do not add to MM |
KAPA Supermix | 25 | 220 |
Klenow Exo Minus | 0.5 | 4.4 |
10uM Orange Primer | 1 | Do not add to MM |
10uM Blue Primer | 1 | Do not add to MM |
Total | 50 | 378.4 |
- 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
File:ZhangLab 2011-06-29 1.JPG
- A1-100 sperm cells
- A2-100 sperm cells (repeat)
- B1-100 ESCs
- B2-100 ESCs (repeat)
- C1-600pg GM12878 gDNA
- D-1.2ng GM12878 gDNA
- E1-NTC
- E2-NTC(MDA)
PAGE Analysis
File:ZhangLab 2 2011-06-29 16hr 00min.jpg
Results
- E1-NTC was amplified and clear bands showed up on the gel image, high possibility of contamination
- Possible source of contamination
- NTC water
- Proteinase K
- E2-NTC added during MDA did not amplify during PCR that shows no contamination was introduced during MDA
To do
- Size Selection on samples A1-E1 (use clone well gel to extract small amount of sample to do cloning)
- Do cloning on size selected A1-E1 samples
- Repeat experiment with only gDNA (UV EB buffer, use RT-PCR grade water as NTC)
- gDNA without proteinase k
- gDNA with proteinase k
- gDNA with protease
- NTC but with proteinase K
- NTC without proteinase K
- gDNA with 0.1ul proteinase K
- Run PCR on 5,8 and 9 with bisulfite verification primers
- Pool A1 all the way to D
- Perform 5 cycles of PCR to add illumina adapter and barcodes