AlanFung:LabNotes/Capturing/CPG97K Low-input/2009-10-9: Difference between revisions
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>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...) |
>Alan6017518 No edit summary |
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*I expect 720,000 cells will have a yield ~3.132ug | *I expect 720,000 cells will have a yield ~3.132ug | ||
Protocol | ==Protocol== | ||
==Sample Preparation== | |||
==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 | |||
{| {{table}} | |||
| align="center" style="background:#f0f0f0;"| | |||
| align="center" style="background:#f0f0f0;"|'''A-2000 cells''' | |||
| align="center" style="background:#f0f0f0;"|'''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) | |||
*98C for 8m | *Centrifuge for 30sec to elute the DNA | ||
*64C for 3.5hr | |||
*4C hold | |||
Revision as of 17:20, 9 October 2009
- 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
Sample Preparation
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
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