AlanFung:Protocol/EZ DNA Methylation-Direct: Difference between revisions

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*Sample Digestion with Proteinase K
*Sample Digestion with Proteinase K
*Bisulfite Conversion of DNA
*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==
==Protocol==
*Sample Preparation
==Sample Preparation==
 
*Adherent cells
 
*Suspension cells


==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
*Sample Digestion with Proteinase K
{| {{table}}
{| {{table}}
| 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 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