Daniel:Notebook/ComboLock/2016-7-25
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Latch 100 and Dextran Test[edit]
Buffer Prep[edit]
Stringent Wash Buffer
Reagent | Stock | Final | Dilution | Amt in 5 mL (uL) |
PBS | 10X | 1X | 10 | 500 uL |
SSC | 20X | 4X | 5 | 1000 uL |
Rnasin | 40000 U/mL | 40 U/mL | 1000 | 5 uL |
nf H2O | NA | NA | NA | 4.5 mL |
LPH Buffer
Reagent | Stock | Final | Dilution | Amt in 1 mL (uL) |
Oligos | 100 uM | 100 nM | 1000 | 100 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 |
CPH Buffer with 10% Dextran Sulfate
Reagent | Stock Conc. | Final Conc. | Amt. Added |
SSC | 20X | 1X | 50 uL |
Tween 20 | 10% | 0.10% | 10 uL |
Rnasin | 40000 U/mL | 40 U/mL | 1 uL |
Riboside Vanadyl Complex | 200 mM | 20 mM | 100 uL |
Poly Vinylsulfonic Acid | 25% | 2.50% | 100 uL |
Salmon Sperm | 10 mg/mL | 100 ug/mL | 10 uL |
Dextran Sulfate | 25% | 10% | 400 uL |
Probes | 100 uM | 100 nM | 1 uL/probe |
Water | NA | NA | 340 uL |
Total | 1 mL |
Wash Buffer Prime (Wash buffer with 2.5% polyvinyl sulfonic acid since it is better at removing free pigment from hematoxylin)
Reagent | Stock | Final | Dilution | Amt in 10 mL (uL) |
PBS | 10X | 1X | 10 | 1000 uL |
Poly Vinylsulfonic Acid | 25% w/v | 2.5% w/v | 10 | 1000 uL |
Tween 20 | 10% | 0.10% | 100 | 100 uL |
Rnasin | 40000 U/mL | 4 U/mL | 10000 | 1 uL |
nf H2O | NA | NA | NA | 7.9 mL |
Protocol[edit]
- C-Probe Hybridization
- Measure cell concentrations in cell counter
- Human - 3.05E5 cells/mL; Mouse - 9.75E5 cells/mL
- Add cells to a new tube such that there are 25000 cells present per sample
- Human- 328 uL (4 samples) uL; Mouse- 26 uL
- Pellet cells via centrifugation at 600g for 3 min
- Wash with 200 uL wash buffer and pellet cells via centrifugation at 600g for 3 min
- Add 100 uL 50% hematoxylin (filtered) and pellet cells via centrifugation at 600g for 3 min
- Wash once with 200 uL wash buffer and pellet cells via centrifugation at 600g for 3 min
- Wash 2 times with 200 uL wash buffer prime and pellet cells via centrifugation at 600g for 3 min
- Pellet cells via centrifugation at 600g for 3 min
- Combine 0.5 uL of each C probe into a 0.2 mL tube with 3 uL nfH2O
- Heat C probes to 90C for 5 min; Chill probes on ice to quench
- Add probes to CPH buffer according to sample matrix, 1 uL probe mix per sample
- 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 100 uL wash buffer by pelleting cells at 600xg for 3 min
- Incubate cells for 20 min at 40C in 150 uL stringent wash buffer
- Resuspend pellet in 100 uL LPH buffer with 100 nM Latch0001
- For sample 3, use 10 uL 100 uM Latch0001 (10 uM final concentration)
- 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 and transfer to a 0.2 mL tube
- 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 | Treatment | |
Sample 1 | X | X | X | None | |
Sample 2 | X | X | X | Dextran | |
Sample 3 | X | X | X | 10X Latch | |
Sample 4 | X | None | |||
Sample 5 | X | X | X | None | |
Sample 6 | X | X | None |