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==Protocol== <ol> <li>Probe Hybridization <ol type="A"> <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> <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> ===Buffers=== <ol type="A"> <li>Insert/backbone Hybridization Buffer</li> <ol type="a"> <li>100 nM insert/backbone oligos</li> <li>1X SSC</li> <li>40 U/mL RNasin</li> <li>PBS</li> </ol> <li>Wash Buffer</li> <ol type="a"> <li>PBS</li> <li>0.1% Tween</li> <li>4 U/mL RNasin</li> </ol>
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