AlanFung:Protocol/EZ DNA Methylation-Direct: Difference between revisions
Jump to navigation
Jump to search
>Alan6017518 |
>Alan6017518 |
||
Line 38: | Line 38: | ||
*Extract 20ul of the supernatant for bisulfite conversion | *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 | *Add 200ul of sample to 130ul of CT conversion reagent solution in a pcr tube | ||
*Vortex the sample to mix | *Vortex the sample to mix |
Revision as of 20:48, 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