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
* 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