Daniel:Protocols/LatchPadlock
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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