AlanFung:LabNotes/Capturing/CPG97K Low-input/2009-10-9

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  • Recovered GM20431 P5 cells
  • Expand up to ~9M cells
  • One CT conversion reagent ~1250ul (not recommended for long term storage)
  • Need about 130ul CT conversion reagent per treatment, hence one tube can use for 9 treatments
  • I am going to aim for the optimal result recommened by loading 80,000 cells per treatment
  • By preparing one CT conversion reagent, I can convert 80,000x9= 720,000 cells
  • from previous experiment 4000 cells gave me 17.4ng CT converted DNA
  • I expect 720,000 cells will have a yield ~3.132ug

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
  • spin down all cells, wash with pbs
  • perform cell count obtain 720,000cells
  • spin down resuspend in 119ul pbs
  • 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 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 to elute the DNA