Daniel:Notebook/ComboLock/2016-6-23: Difference between revisions
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<li>Put 1 uL C1 Probe and 1 uL C2 probe into a 0.2 mL tube</li> | <li>Put 1 uL C1 Probe and 1 uL C2 probe into a 0.2 mL tube</li> | ||
<li>Heat C probes to 90C for 5 min; Chill probes on ice to quench</li> | <li>Heat C probes to 90C for 5 min; Chill probes on ice to quench</li> | ||
<li>Add probes to | <li>Add probes to CPH buffer for final concentration of 100 nM (Add 1 uL probe mixture to 0.5mL hybridization buffer)</li> | ||
<li>Incubate probes at 40C for 1 hour with vigorous agitation</li> | <li>Incubate probes at 40C for 1 hour with vigorous agitation</li> | ||
<li>Wash three times with 200 uL wash buffer and pelleting at 600xg for 3 min</li> | <li>Wash three times with 200 uL wash buffer and pelleting at 600xg for 3 min</li> | ||
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<ol type="A"> | <ol type="A"> | ||
<li>Pellet at 600xg for 3 min and remove supernatant</li> | <li>Pellet at 600xg for 3 min and remove supernatant</li> | ||
<li>Resuspend pellet in 50 uL | <li>Resuspend pellet in 50 uL LPH buffer with 100 nM insert/backbone oligos for 30 min at 37C</li> | ||
<li>Wash twice</li> | <li>Wash twice with 200 uL wash buffer</li> | ||
<ol type="a"><li>Wash by pelleting cells at 600xg for 3 min</li></ol></ol> | <ol type="a"><li>Wash by pelleting cells at 600xg for 3 min</li></ol></ol> | ||
<li>Circularization</li> | <li>Circularization</li> | ||
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<ol type="A"> | <ol type="A"> | ||
<li>C-Probe ( | <li>C-Probe Hybridization (CPH) Buffer</li> | ||
<ol type="a"> | <ol type="a"> | ||
<li>100 nM insert/backbone oligos</li> | <li>100 nM insert/backbone oligos</li> | ||
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<li>4 U/mL RNasin</li> | <li>4 U/mL RNasin</li> | ||
</ol> | </ol> | ||
<li>Latch-Padlock Hybridization (LPH) Buffer</li> |
Revision as of 22:58, 22 June 2016
Stage 2 Test
The purpose of this experiment is to test the latch and padlock binding, termed stage 2 (at the moment). This is based on the success of the VIM binding for both C-Probes and antibodies.
Protocol
Sample Matrix
Sample | VIM-C1 (1 uL) | VIM-C2 (1 uL) |
Sample 1 | X | X |
Sample 2 |
- C-Probe Hybridization
- Split cells into 2 samples (1 mL methanol each)
- Pellet cells via centrifugation at 600g for 3 min
- Put 1 uL C1 Probe and 1 uL C2 probe into a 0.2 mL tube
- Heat C probes to 90C for 5 min; Chill probes on ice to quench
- Add probes to CPH buffer for final concentration of 100 nM (Add 1 uL probe mixture to 0.5mL hybridization buffer)
- Incubate probes at 40C for 1 hour with vigorous agitation
- Wash three times with 200 uL wash buffer and pelleting at 600xg for 3 min
- Incubate cells for 20 min at 40C in 200 uL stringent wash buffer
- Latch and Padlock Hybridization
- Pellet at 600xg for 3 min and remove supernatant
- Resuspend pellet in 50 uL LPH buffer with 100 nM insert/backbone oligos for 30 min at 37C
- Wash twice with 200 uL wash buffer
- 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 overnight for 4-20 hours
- Continued tomorrow
- C-Probe Hybridization (CPH) Buffer
- 100 nM insert/backbone oligos
- 1X SSC
- 40 U/mL RNasin
- PBS
- Wash Buffer
- PBS
- 0.1% Tween
- 4 U/mL RNasin
- Latch-Padlock Hybridization (LPH) Buffer