AlanFung:Protocol/EZ DNA Methylation-Direct: Difference between revisions
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[[Media:EZ_DNA_Methylation_Direct_Kit_Protocol.pdf|EZ_DNA_Methylation_Direct_Kit_Protocol PDF]] | [[Media:EZ_DNA_Methylation_Direct_Kit_Protocol.pdf|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 | |||
{| {{table}} | |||
| align="center" style="background:#f0f0f0;"| | |||
| align="center" style="background:#f0f0f0;"|'''A-2000 cells''' | |||
| align="center" style="background:#f0f0f0;"|'''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 20ul 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 | |||
*Add 200ul of M-Wash buffer to the column and repeat centrifuge | |||
*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 |
Latest revision as of 23:01, 29 January 2010
Draft
EZ_DNA_Methylation_Direct_Kit_Protocol PDF
Overview[edit]
- Sample preparation
- Sample Digestion with Proteinase K
- Bisulfite Conversion of DNA
Reagent Preparation[edit]
Proteinase K[edit]
- 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[edit]
- 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[edit]
- 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[edit]
Sample Preparation[edit]
For optimal results use 8x10^4 cells per treatment[edit]
- 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[edit]
- Add 20ul 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
- Add 200ul of M-Wash buffer to the column and repeat centrifuge
- 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