Daniel:Protocols/CProbeLigate: Difference between revisions

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<li>Hybridization</li>
<li>Hybridization</li>
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<ol type="A">
<li>Pellet cells via centrifugation at 600g for 3 min</li>
<li>Pellet cells via centrifugation at 600g for 3 min</li>
<li>Heat C probes to 90C for 5 min; Chill probes on ice to quench</li>
<li>Heat C probes to 90C for 5 min; Chill probes on ice to quench</li>
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==Buffers==
 
'''Wash Buffer'''
'''Wash Buffer'''
*PBS
*PBS

Latest revision as of 21:20, 6 May 2016

C Probe Hybridization[edit]

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

Protocol[edit]

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[edit]

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