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
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 following the sample matrix Sample Matrix
- Wash twice with 200 uL wash buffer
- Wash by pelleting cells at 600xg for 3 min
- Resuspend with 20 uL 1x Amp Ligase buffer
- Circularization
- Prepare 10 uL KLN mix
- Add 2 uL KLN mix to 20 uL of reaction
- Incubate at 55C overnight (XX 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
- 100 nM insert/backbone oligos
- 1X SSC
- 40 U/mL RNasin
- PBS
- KLN Mix 10 uL total
- 2 uL HemoKlentaq (20% v/v)
- 1 U/μL Ampligase
- 1 uL 1 mM dNTP mix (100 μM ea)
- Add 10 uL 10mM dNTP mix to 90 uL nfH2O
- 1 uL 1x Ampligase Buffer 1x
- 6 uL nfH2O
Batch | Cell Sample | Latch (1 uL) | Padlock (1 uL) |
Batch 1 | S1 | X | X |
Batch 2 | S1 | X | |
Batch 3 | S1 | X | |
Batch 4 | S2 | X | X |