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

From ZhangLabWiki
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
===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