Daniel:Protocols/CProbeLigate
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C Probe Hybridization
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
- Fix cells
- Pellet cells at 600g for 3 min and resuspend at concentration of ~1,000,000/mL
- Fix cells in medium with 1.6% paraformaldehyde for 10 min at RT
- Wash cells with wash buffer
- Pellet cells and permeabilize with ice-cold methanol for 10 min on ice Once in methanol cells may be stored long term
- Hybridization
- Pellet cells via centrifugation at 600g for 3 min
- Heat C probes to 90C for 5 min; Chill probes on ice to quench
- Add probes to cells for final concentration of 100 nM
- Incubate probes at 40C for 1 hour with vigorous agitation
- Wash three times with wash buffer and pelleting at 600xg for 3 min
- Incubate cells for 20 min at 40C in stringent wash buffer
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