Daniel:Protocols/CProbeLigate: Difference between revisions

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(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>Fix cells</li>
<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 2X SSC buffer</li>
<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

Back to Notebook

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
  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


Wash Buffer

  • PBS
  • 0.1% Tween
  • 4 U/mL RNasin