Daniel:Protocols/CProbeLigate: Difference between revisions
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>Djacobse (Created page with "=C Probe Hybridization= Back to Notebook This page describes the protocol for hybridizing C probes as used in PLAYR Design (as from [http://www.ncbi.nlm....") |
>Djacobse |
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<ol> | <ol> | ||
<li>Fix cells</li> | |||
<ol type="A"> | <ol type="A"> | ||
<li>Pellet cells at 600g for 3 min and resuspend at concentration of ~1,000,000/mL</li> | |||
<li>Pellet cells and resuspend at concentration of ~1,000,000/mL</li> | |||
<li>Fix cells in medium with 1.6% paraformaldehyde for 10 min at RT</li> | <li>Fix cells in medium with 1.6% paraformaldehyde for 10 min at RT</li> | ||
<li>Wash cells with | <li>Wash cells with wash buffer</li> | ||
<li>Pellet cells and permeabilize with ice-cold methanol for 10 min on ice</li> | <li>Pellet cells and permeabilize with ice-cold methanol for 10 min on ice</li> | ||
Once in methanol cells may be stored long term | |||
</ol> | |||
<li>Hybridization</li> | |||
<ol type-"A"> | |||
<li> | |||
</ol> | </ol> | ||
</ol> | </ol> | ||
'''Wash Buffer''' | |||
*PBS | |||
*0.1% Tween | |||
*4 U/mL RNasin |
Revision as of 23:26, 5 May 2016
C Probe Hybridization
This page describes the protocol for hybridizing C probes as used in PLAYR Design (as from Nolan lab).
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
Wash Buffer
- PBS
- 0.1% Tween
- 4 U/mL RNasin