Daniel:Notebook/ComboLock/2016-5-23

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Revision as of 17:03, 23 May 2016 by >Djacobse (→‎C Probe Hybridization Test)
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C Probe Hybridization Test

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This will be the first test of the C Probe hybridization protocol for Combo-Lock. I have been growing U87MG cells for several days now and am ready to passage them. When I passage I'll take the remaining 90% for this test. Since I don't have the antibodies ready yet (I am waiting on some columns) I am just testing the C Probes.

Protocol

Base Protocol

  1. Fix cells
    1. Start with confluent U87MG cells
    2. Remove media (EMEM) and wash with 1X PBS
    3. Trypsinize cells using 0.5 mL trypsin LE express; incubate 5 min at 37C
    4. Resuspend cells in by adding 4.5 mL media; use 0.5 mL to passage
    5. Pellet remainder of the cells in a 15 mL Falcon tube at 600g for 3 min
    6. Remove supernatant
    7. Fix cells in 5 mL EMEM with 1.6% paraformaldehyde for 10 min at RT
    8. Pellet cells by centrifuging at 600xg for 3 min
    9. Wash cells with wash buffer
    10. Pellet cells and permeabilize with ice-cold methanol for 10 min on ice
    11. Once in methanol cells may be stored long term
  2. Hybridization
    1. Pellet cells via centrifugation at 600g for 3 min
    2. Heat C probes to 90C for 5 min; Chill probes on ice to quench
    3. Add probes to cells for final concentration of 100 nM
    4. Incubate probes at 40C for 1 hour with vigorous agitation
    5. Wash three times with wash buffer and pelleting at 600xg for 3 min
    6. Incubate cells for 20 min at 40C in stringent wash buffer

Buffers

Wash Buffer
PBS
0.1% Tween-20
4 U/mL RNasin