Daniel:Protocols/LatchPadlock
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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:
- 20% v/v HemoKlentaq
- 0.5 U/μL Ampligase
- 100 μM of dNTP mix
- 1x Ampligase Buffer
- 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