AlanFung:LabNotes/Capturing/BisFirstExon 2009 12 2: Difference between revisions
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*Add 260ul(D5020) or 1040ul(D5021) of proteinase K storage buffer to the tube containing proteinase K | *Add 260ul(D5020) or 1040ul(D5021) of proteinase K storage buffer to the tube containing proteinase K | ||
*Dissolve completely and store at -20C | *Dissolve completely and store at -20C | ||
===CT Conversion Reagent=== | ===CT Conversion Reagent (1250ul Total good for 9rxns)=== | ||
*CT Conversion Reagent is light sensitive, so minize its exposure to light | *CT Conversion Reagent is light sensitive, so minize its exposure to light | ||
*Add 790ul M-solubilization buffer and 300ul M-Dilution Buffer to a tube of CT Conversion Reagent | *Add 790ul M-solubilization buffer and 300ul M-Dilution Buffer to a tube of CT Conversion Reagent | ||
*Mix by frequent vortexing at RT for 10m | *Mix by frequent vortexing at RT for 10m | ||
*Mix 160ul of M-Reaction Buffer and mix an additional 1m | *Mix 160ul of M-Reaction Buffer and mix an additional 1m | ||
===M-Wash Buffer=== | ===M-Wash Buffer=== | ||
*Add 24ml of 100% ethanol to the 6ml M-Wash Buffer concentrate (D5020)before use | *Add 24ml of 100% ethanol to the 6ml M-Wash Buffer concentrate (D5020)before use |
Revision as of 23:13, 2 December 2009
Bisulfite Conversion of Sample NA12878,NA18507, NA20431, CV-Fibr, CV-iPS-B, CV-iPS-F
Overview
- Sample preparation
- Sample Digestion with Proteinase K
- Bisulfite Conversion of DNA
Sample
NA12878 (160ng/ul) NA18507 (257ng/ul) NA20431 (314ng/ul) CV-Fibr (712ng/ul) CV-iPS-B (358ng/ul) CV-iPS-F (309ng/ul)
Sample Preparation
Sample Prep | ng/ul | To Get 1000ng | Make it up to 40ul |
NA12878 | 160.000 | 6.250 | 33.750 |
NA18507 | 257.000 | 3.891 | 36.109 |
NA20431 | 314.000 | 3.185 | 36.815 |
CV-Fibr | 712.000 | 1.404 | 38.596 |
CV-iPS-B | 358.000 | 2.793 | 37.207 |
CV-iPS-F | 309.000 | 3.236 | 36.764 |
Reagent Preparation
Proteinase K
- Add 260ul(D5020) or 1040ul(D5021) of proteinase K storage buffer to the tube containing proteinase K
- Dissolve completely and store at -20C
CT Conversion Reagent (1250ul Total good for 9rxns)
- CT Conversion Reagent is light sensitive, so minize its exposure to light
- Add 790ul M-solubilization buffer and 300ul M-Dilution Buffer to a tube of CT Conversion Reagent
- Mix by frequent vortexing at RT for 10m
- Mix 160ul of M-Reaction Buffer and mix an additional 1m
M-Wash Buffer
- Add 24ml of 100% ethanol to the 6ml M-Wash Buffer concentrate (D5020)before use
- Add 96ml 100% ethanol to the 24ml M-Wash buffer concentrate (D5021) before use
Protocol
Sample Preparation
For optimal results use 500ng of DNA per treatment
- Turn on heat bath to 50C before sample prep
- Wash cells with PBS once and perform a cell count
- Sample Digestion with Proteinase K
A-2000 cells | B-100,000 cells | |
M-Digestion Buffer (2X) | 10ul | 13ul |
Sample | up to 9ul | up to 12ul |
Proteinase K | 1ul | 1ul |
H20 | X ul | x ul |
Total Volume | 20ul | 26 ul |
- Incubate sample(s) for 20m at 50C
- If following procedure A, procedd directly to Section II
- If following procedure B, mix content throughly with vortex then centrifuge for 5m at 10,000g
- Extract 20ul of the supernatant for bisulfite conversion
Section II Bisulfite conversion of DNA
- Add 200ul of sample to 130ul of CT conversion reagent solution in a pcr tube
- Vortex the sample to mix
- Pulse centrifuge
- Perform
*98C for 8m *64C for 3.5hr *4C hold
- Add 600ul of M binding buffer into a column assembly
- Load sample(s) to the column
- Close the cap and mix by inverting the column several times
- Centrifuge at >10,000g for 30sec
- Discard the flow through
- Add 100ul of M-Wash Buffer to the column
- Centrifuge at full speed for 30sec
- Add 200ul of M-Desulphonation buffer to the column and let stand at RT for 20m
- Centrifuge for 30sec
- Place the column into a 1.5ml tube
- Add 10ul of M-elution buffer directly to the column matrix (Volume can be adjusted, depending on the requirements)
- Centrifuge for 30sec to elute the DNA