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

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Revision as of 17:19, 9 October 2009 by >Alan6017518 (New page: *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...)
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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

   * 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 

[edit] 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 to elute the DNA