Daniel:Protocols/LatchPadlock: Difference between revisions
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>Djacobse (Created page with "=Latch and Padlock Hybridization= Back to Notebook ==Protocol== <ol> <li>Start from hybridized C Probes or [[Daniel:Pr...") |
>Djacobse |
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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=" | <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> | ||
Line 23: | Line 53: | ||
<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]
Protocol[edit]
- Probe Hybridization
- Start from hybridized C Probes or antibodies
- Make sure probes have been washed as per the protocol
- After washing, incubate with 100 nM insert/backbone oligos for 30 min at 37C
- Alternative Protocol
- Start with 94C for 30 seconds
- Decrease to 55C at a rate of 0.02C/sec
- Hold at 55C for 20 hours
- Wash twice
- Wash by pelleting cells at 600xg for 3 min
- Circularization
- Prepare KLN mix with:
- 20% v/v HemoKlentaq
- 0.5 U/μL Ampligase
- 100 μM of dNTP mix
- 1x Ampligase Buffer
- Add 2 uL KLN mix to 20 uL of reaction
- Incubate at 55C for 4-20 hours
- Heat inactivate enzyme by incubating for 2 minutes at 94C
- Exonuclease Digestion
- Prepare exonuclease I/III mix by mixing exonuclease I (20 units/μL) and exonuclease III (200 units/μL) in 1:1 ratio
- Add 2 μL of exonuclease I/III mix to reaction
- Mix the reaction by swirling pipette around the well 5 times
- Incubate reaction at 37 ºC for 2 hours
- Heat inactivate enzyme by incubating at 94C for 5 minutes
Buffers[edit]
- Insert/backbone Hybridization Buffer
- 100 nM insert/backbone oligos
- 1X SSC
- 40 U/mL RNasin
- PBS
- Wash Buffer
- PBS
- 0.1% Tween
- 4 U/mL RNasin