Daniel:Protocols/LatchPadlock: Difference between revisions

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>Djacobse
>Djacobse
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</ol>
</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></ol>
<li>Circularization</li>
<li>Circularization</li>
<ol type="A">
<ol type="A">
<li>Setup up reaction well with:</li>
<li>Setup up reaction well with:</li>
<ol type="a">
<ol type="a">
<li>100-200ng DNA (cell amount?)</li>
<li>20% v/v HemoKlentaq</li>
<li>Normalized amount of latch
<li>0.5 U/μL Ampligase</li>
<li>100 μM of dNTP mix</li>
<li>1x Ampligase Buffer</li>
</ol>
</ol>
</ol>
</ol>

Revision as of 22:58, 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
  2. Circularization
    1. Setup up reaction well with:
      1. 20% v/v HemoKlentaq
      2. 0.5 U/μL Ampligase
      3. 100 μM of dNTP mix
      4. 1x Ampligase Buffer


    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