AlanFung:Protocol/EZ DNA Methylation-Direct: Difference between revisions
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| align="center" style="background:#f0f0f0;"| | | align="center" style="background:#f0f0f0;"| | ||
| align="center" style="background:#f0f0f0;"|'''2000 cells''' | | align="center" style="background:#f0f0f0;"|'''A-2000 cells''' | ||
| align="center" style="background:#f0f0f0;"|'''100,000 cells''' | | align="center" style="background:#f0f0f0;"|'''B-100,000 cells''' | ||
|- | |- | ||
| M-Digestion Buffer (2X)||10ul||13ul | | M-Digestion Buffer (2X)||10ul||13ul | ||
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*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 | |||
* |
Revision as of 20:41, 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