AlanFung:LabNotes/CTCF/2011-6-30: Difference between revisions
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>Alan6017518 (→MDA) |
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| align="center" style="background:#f0f0f0;"|'''MM''' | | align="center" style="background:#f0f0f0;"|'''MM''' | ||
|- | |- | ||
| 10X RepliPhi Buffer||2|| | | 10X RepliPhi Buffer||2||13.2 | ||
|- | |- | ||
| 1mM N9 Primer||1|| | | 1mM N9 Primer||1||6.6 | ||
|- | |- | ||
| 25mM dNTP||0.8|| | | 25mM dNTP||0.8||5.28 | ||
|- | |- | ||
| RepliPhi Phi29 (100U/ul)||1|| | | RepliPhi Phi29 (100U/ul)||1||6.6 | ||
|- | |- | ||
| Klenow Exo Minus (10U/ul)||0.5||3. | | Klenow Exo Minus (10U/ul)||0.5||3.3 | ||
|- | |- | ||
| H2O||4.7|| | | H2O||4.7||31.02 | ||
|- | |- | ||
| Total||10|| | | Total||10||66 | ||
|} | |} | ||
*Add 10ul of MDA mastermix to each sample | *Add 10ul of MDA mastermix to each sample | ||
Line 119: | Line 119: | ||
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 |
Revision as of 23:45, 1 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
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