Daniel:Notebook/ComboLock/2016-6-23
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Stage 2 Test[edit]
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[edit]
Prep[edit]
Wash Buffer
Reagent | Stock | Final | Dilution | Amt in 10 mL |
PBS | 10X | 1X | 10 | 1 mL |
Tween 20 | 100% | 0.10% | 1000 | 10 uL |
Rnasin | 40000 U/mL | 4 U/mL | 1000 | 10 uL |
nf H2O | NA | NA | NA | 9 mL |
Stringent Wash Buffer
Reagent | Stock | Final | Dilution | Amt in 1 mL (uL) |
PBS | 10X | 1X | 10 | 100 uL |
SSC | 20X | 4X | 5 | 200 uL |
Rnasin | 40000 U/mL | 40 U/mL | 100 | 10 uL |
nf H2O | NA | NA | NA | 690 uL |
LPH Buffer
Reagent | Stock | Final | Dilution | Amt in 100 uL (uL) |
Oligos | 10 uM | 100 nM | 100 | 1 uL |
SSC | 20X | 1X | 5 | 20 uL |
Rnasin | 40000 U/mL | 40 U/mL | 100 | 1 uL |
PBS | 10X | 1X | 10 | 10 uL |
nf H2O | NA | NA | NA | 68 uL |
Experiment[edit]
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
- Combine 1.5 uL of each C1 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 2 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 100 uL LPH buffer per sample (600 uL S1, 200 uL S2)
- Add 1 uL of each insert/backbone (final conc. 100 nM each) Sample Matrix
- Incubate for 30 min at 37C following the 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 20 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 20 uL total
- 4 uL HemoKlentaq (20% v/v)
- 2 μL Ampligase (0.5 U/uL)
- 2 uL 1 mM dNTP mix (100 μM ea)
- Add 10 uL 10mM dNTP mix to 90 uL nfH2O
- 2 uL 1x Ampligase Buffer 1x
- 10 uL nfH2O
Batch | Cell Sample | Latch0001 (1 uL) | Latch0002 (1 uL) | Padlock0001-48 (1 uL) | Padlock0001-68 (1 uL) | Padlock0001-88 (1 uL) |
Batch 1 | S1 | X | X | |||
Batch 2 | S1 | X | X | |||
Batch 3 | S1 | X | X | |||
Batch 4 | S1 | X | X | |||
Batch 5 | S1 | X | X | |||
Batch 6 | S1 | X | X | |||
Batch 7 | S2 | X | X | |||
Batch 8 | S2 | X | X |