AlanFung:LabNotes/CTCF/2011-6-30: Difference between revisions
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>Alan6017518 (→To do) |
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(29 intermediate revisions by the same user not shown) | |||
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|| | | 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 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 | ||
*Add 0.1X 3M NaOAC | *Add 0.1X 3M NaOAC | ||
*Add 1ul glycoblue | *Add 1ul glycoblue | ||
*Store at -80C for 30min (CONTINUE ON | *Store at -80C for 30min (CONTINUE ON 7/5) | ||
*Spin down at 10,000rpm at 4C for 15min | *Spin down at 10,000rpm at 4C for 15min | ||
*Remove all liquid | *Remove all liquid | ||
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
- 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[edit]
- Proteinase K/Protease Digestion
- Bisulfite Conversion
- Heat Denature
- MDA
- Ethanol Precipitation
- Nextera Library Construction (Low input protocol)
- 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
- 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[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
- 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[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
- 96C, 3m
- 95C, 30s
- 62C, 1m
- 72C, 1m
- Go to step2 repeat 39 times
- 72C, 5m
- 4C, Forever
Page Analysis[edit]
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
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