Daniel:Protocols/LatchPadlock: Difference between revisions

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(Created page with "=Latch and Padlock Hybridization= Back to Notebook ==Protocol== <ol> <li>Start from hybridized C Probes or [[Daniel:Pr...")
 
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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>
<ol type="a"><li>Wash by pelleting cells at 600xg for 3 min</li></ol></ol>
<li>Circularization</li>
<ol type="A">
<li>Prepare KLN mix with:</li>
<ol type="a">
<li>20% v/v HemoKlentaq</li>
<li>0.5 U/μL Ampligase</li>
<li>100 μM of dNTP mix</li>
<li>1x Ampligase Buffer</li>
</ol>
<li>Add 2 uL KLN mix to 20 uL of reaction</li>
<li>Incubate at 55C for 4-20 hours</li>
<li>Heat inactivate enzyme by incubating for 2 minutes at 94C</li>
</ol>
<li>Exonuclease Digestion</li>
<ol type="A">
<li>Prepare exonuclease I/III mix by mixing exonuclease I (20 units/μL) and exonuclease III (200 units/μL) in 1:1 ratio</li>
<li>Add 2 μL of exonuclease I/III mix to reaction</li>
<li>Mix the reaction by swirling pipette around the well 5 times</li>
<li>Incubate reaction at 37 ºC for 2 hours</li>
<li>Heat inactivate enzyme by incubating at 94C for 5 minutes</li>
</ol>
</ol>
</ol>


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<li>PBS</li>
<li>PBS</li>
</ol>
</ol>
<li>Wash Buffer</li>
<ol type="a">
<li>PBS</li>
<li>0.1% Tween</li>
<li>4 U/mL RNasin</li>
</ol>
</ol>

Latest revision as of 23:06, 11 June 2016

Latch and Padlock Hybridization[edit]

Back to Notebook

Protocol[edit]

  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. Prepare KLN mix with:
      1. 20% v/v HemoKlentaq
      2. 0.5 U/μL Ampligase
      3. 100 μM of dNTP mix
      4. 1x Ampligase Buffer
    2. Add 2 uL KLN mix to 20 uL of reaction
    3. Incubate at 55C for 4-20 hours
    4. Heat inactivate enzyme by incubating for 2 minutes at 94C
  3. Exonuclease Digestion
    1. Prepare exonuclease I/III mix by mixing exonuclease I (20 units/μL) and exonuclease III (200 units/μL) in 1:1 ratio
    2. Add 2 μL of exonuclease I/III mix to reaction
    3. Mix the reaction by swirling pipette around the well 5 times
    4. Incubate reaction at 37 ºC for 2 hours
    5. Heat inactivate enzyme by incubating at 94C for 5 minutes


Buffers[edit]

  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