Daniel:Protocols/CProbeLigate

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Revision as of 23:42, 5 May 2016 by >Djacobse (→‎Protocol)
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C Probe Hybridization

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This page describes the protocol for hybridizing C probes as used in PLAYR Design (as from Nolan lab paper).

Protocol

Resuspend oligos in nuclease free water to concentration of 100 uM
  1. Fix cells
    1. Pellet cells at 600g for 3 min and resuspend at concentration of ~1,000,000/mL
    2. Fix cells in medium with 1.6% paraformaldehyde for 10 min at RT
    3. Wash cells with wash buffer
    4. Pellet cells and permeabilize with ice-cold methanol for 10 min on ice
    5. 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
  • 4 U/mL RNasin

Stringent Wash Buffer

  • PBS
  • 4X SSC
  • 40 U/mL RNasin

Hybridization Buffer

  • 1X SSC
  • 2.5% vol/vol polyvinylsulfonic acid
  • 20 mM ribonucleoside vanadyl complex
  • 40 U/mL RNasin
  • 0.1% Tween
  • 100 ug/mL salmon sperm