AlanFung:Protocol/EZ DNA Methylation-Direct: Difference between revisions
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*4C hold | *4C hold | ||
*Add 600ul of M binding buffer into a column assembly | *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 |
Revision as of 20:47, 14 August 2009
Draft
EZ_DNA_Methylation_Direct_Kit_Protocol PDF
Overview
- Sample preparation
- Sample Digestion with Proteinase K
- Bisulfite Conversion of DNA
Protocol
- Sample Preparation
- Adherent cells
- Suspension cells
- 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