Daniel:Protocols/LatchPadlock: Difference between revisions

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<ol>
<ol>
<li>Probe Hybridization
<ol type="A">
<li>Start from hybridized [[Daniel:Protocols/CProbeLigate|C Probes]] or [[Daniel:Protocols/AbHybridization|antibodies]]</li>
<li>Start from hybridized [[Daniel:Protocols/CProbeLigate|C Probes]] or [[Daniel:Protocols/AbHybridization|antibodies]]</li>
<ol type="A"><li>Make sure probes have been washed as per the protocol</li></ol>
<ol type="a"><li>Make sure probes have been washed as per the protocol</li></ol>
<li>After washing, incubate with 100 nM insert/backbone oligos for 30 min at 37C</li>
<li>After washing, incubate with 100 nM insert/backbone oligos for 30 min at 37C</li>
<ol type="a">
<li>'''Alternative Protocol'''</li>
<li>Start with 94C for 30 seconds</li>
<li>Decrease to 55C at a rate of 0.02C/sec</li>
<li>Hold at 55C for 20 hours</li>
</ol>
<li>Wash twice</li>
<li>Wash twice</li>
<ol type="A"><li>Wash by pelleting cells at 600xg for 3 min</li></ol>
<ol type="a"><li>Wash by pelleting cells at 600xg for 3 min</li></ol>
 
<li>Circularization</li>
<ol type="A">
<li>Setup up reaction well with:</li>
<ol type="a">
<li>100-200ng DNA (cell amount?)</li>
<li>Normalized amount of latch
</ol>
</ol>
</ol>



Revision as of 22:56, 11 June 2016

Latch and Padlock Hybridization

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Protocol

  1. Probe Hybridization
    1. Start from hybridized C Probes or antibodies
      1. Make sure probes have been washed as per the protocol
    2. After washing, incubate with 100 nM insert/backbone oligos for 30 min at 37C
      1. Alternative Protocol
      2. Start with 94C for 30 seconds
      3. Decrease to 55C at a rate of 0.02C/sec
      4. Hold at 55C for 20 hours
    3. Wash twice
      1. Wash by pelleting cells at 600xg for 3 min
    4. Circularization
      1. Setup up reaction well with:
        1. 100-200ng DNA (cell amount?)
        2. Normalized amount of latch


      Buffers

      1. Insert/backbone Hybridization Buffer
        1. 100 nM insert/backbone oligos
        2. 1X SSC
        3. 40 U/mL RNasin
        4. PBS
      2. Wash Buffer
        1. PBS
        2. 0.1% Tween
        3. 4 U/mL RNasin