AlanFung:Protocol/EZ DNA Methylation-Direct: Difference between revisions
Jump to navigation
Jump to search
>Alan6017518 |
>Alan6017518 |
||
Line 22: | Line 22: | ||
==Protocol== | ==Protocol== | ||
===Sample Preparation=== | ===Sample Preparation=== | ||
==For optimal results use 8x10^4 cells per treatment== | |||
*Turn on heat bath to 50C before sample prep | *Turn on heat bath to 50C before sample prep | ||
*Wash cells with PBS once and perform a cell count | *Wash cells with PBS once and perform a cell count |
Revision as of 17:04, 9 October 2009
Draft
EZ_DNA_Methylation_Direct_Kit_Protocol PDF
Overview
- Sample preparation
- Sample Digestion with Proteinase K
- Bisulfite Conversion of DNA
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
- 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 8x10^4 cells 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