Daniel:Notebook/ComboLock/2016-6-23
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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
- C-Probe Hybridization
- Pellet cells via centrifugation at 600g for 3 min
- Heat C probes to 90C for 5 min; Chill probes on ice to quench
- Add probes to cells for final concentration of 100 nM
- Incubate probes at 40C for 1 hour with vigorous agitation
- Wash three times with wash buffer and pelleting at 600xg for 3 min
- Incubate cells for 20 min at 40C in stringent wash buffer
- Latch and Padlock Hybridization
- Start from hybridized C Probes or antibodies
- 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