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
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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==
[edit] Sample Preparation
==Sample Preparation==
[edit] For optimal results use 8x10^4 cells per treatment


    * Turn on heat bath to 50C before sample prep
==For optimal results use 8x10^4 cells per treatment==
    * Wash cells with PBS once and perform a cell count
*Turn on heat bath to 50C before sample prep
    * Sample Digestion with Proteinase K  
*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


A-2000 cells B-100,000 cells
===Section II Bisulfite conversion of DNA===
M-Digestion Buffer (2X) 10ul 13ul
*Add 200ul of sample to 130ul of CT conversion reagent solution in a pcr tube
Sample up to 9ul up to 12ul
*Vortex the sample to mix
Proteinase K 1ul 1ul
*Pulse centrifuge  
H20 X ul x ul
*Perform  
Total Volume 20ul 26 ul
*98C for 8m
 
*64C for 3.5hr
    * Incubate sample(s) for 20m at 50C
*4C hold
    * If following procedure A, procedd directly to Section II
*Add 600ul of M binding buffer into a column assembly  
    * If following procedure B, mix content throughly with vortex then centrifuge for 5m at 10,000g
*Load sample(s) to the column
    * Extract 20ul of the supernatant for bisulfite conversion
*Close the cap and mix by inverting the column several times
 
*Centrifuge at >10,000g for 30sec
[edit] Section II Bisulfite conversion of DNA
*Discard the flow through
 
*Add 100ul of M-Wash Buffer to the column
    * Add 200ul of sample to 130ul of CT conversion reagent solution in a pcr tube
*Centrifuge at full speed for 30sec
    * Vortex the sample to mix
*Add 200ul of M-Desulphonation buffer to the column and let stand at RT for 20m
    * Pulse centrifuge
*Centrifuge for 30sec
    * Perform  
*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
 
    * 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

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