Daniel:Notebook/ComboLock/2016-7-18
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C Probe Test 3-SOD1/VIM-Stringent Washes[edit]
Protocol[edit]
Buffer Prep[edit]
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 | 1000 | 1 uL |
nf H2O | NA | NA | NA | 700 uL |
LPH Buffer
Reagent | Stock | Final | Dilution | Amt in 1 mL (uL) |
Oligos | 100 uM | 100 nM | 1000 | 1 uL |
SSC | 20X | 1X | 20 | 50 uL |
Rnasin | 40000 U/mL | 40 U/mL | 1000 | 1 uL |
PBS | 10X | 1X | 10 | 100 uL |
nf H2O | NA | NA | NA | 850 uL |
KLN Mix
Reagent | Stock | uL added |
Amp Ligase Buffer | 10X | 2 |
dNTPs | 100 uM | 2 |
Hemo Klentaq | NA | 4 |
Amp Ligase | 5 U/uL | 2 |
nfH2O | NA | 10 |
Resuspended SOD1 C probes in 40 uL nfH2O each (100 uM)
Experiment[edit]
- C-Probe Hybridization
- Add cells to a new tube such that there are 100000 cells present
- Human-500 uL; Mouse-140 uL
- Pellet cells via centrifugation at 600g for 3 min
- Combine 1.5 uL of each C 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 with 100 nM Padlock0001-68
- Incubate for 30 min at 37C
- Wash twice with 200 uL wash buffer by pelleting cells at 600xg for 3 min
- Incubate cells for 20 min at 40C in 200 uL stringent wash buffer
- Resuspend pellet in 100 uL LPH buffer with 100 nM Latch0001
- Incubate for 30 min at 37C
- Wash twice with 200 uL wash buffer by pelleting cells at 600xg for 3 min
- Incubate cells for 20 min at 40C in 200 uL stringent wash buffer
- 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 Probes-VIM (C1+C2) | C Probes-SOD1 (C1+C2) | U87MG | 3T3 Cells | |
Sample 1 | X | X | X | |
Sample 2 | X | |||
Sample 3 | X | X | X | |
Sample 4 | X | |||
Sample 5 | X | X |