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=" | <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=" | <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
Protocol
- 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
- Setup up reaction well with:
- 100-200ng DNA (cell amount?)
- Normalized amount of latch
- 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
Buffers