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 hybridization buffer for final concentration of 100 nM (0.5 uL/probe/sample using 0.5mL hybridization buffer)
- 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
- Pellet at 600xg for 3 min
- Incubate with 100 nM insert/backbone oligos for 30 min at 37C
- 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
- qPCR
- Set up reaction according to table
- qPCR Cycles
- 95C 3 min
- 95C 3 sec
- 55C 30 sec
- 72C 20 sec
- plate read
- goto b x20
- 72C 2 min
- 16C hold