Daniel:Notebook/ComboLock/2016-7-11
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Million Fold Test[edit]
After conversations with Dr. Zhang and Andrew on Friday I'm going to try a SOD1 test using 5X and 10X amounts from the original protocol.
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 | 100 | 10 uL |
nf H2O | NA | NA | NA | 690 uL |
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 |
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 |
Wash Buffer
Reagent | Stock Conc. | Final Conc. | Amt. Added |
PBS | 10X | 1X | 5 mL |
Tween 20 | 100% | 0.10% | 50 uL |
Rnasin | 40000 U/mL | 4 U/mL | 5 uL |
Water | NA | NA | 44.9 mL |
Total Volume | 50 mL |
Store in 5 mL aliquots
Experiment[edit]
- C-Probe Hybridization
- Measure cell concentrations in cell counter
- Human - cells/mL; Mouse - cells/mL
- Add cells to a new tube such that there are 100000 cells present
- Human- uL; Mouse- uL
- Pellet cells via centrifugation at 600g for 3 min
- Combine 17 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 according to sample matrix
- 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 200 nM Padlock0001-68
- 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 pellet in 100 uL LPH buffer with 1 uM Latch0001
- 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
Sample | Cell Type | uL 100 uM C Probe | Final C Probe Concentration | Final C Probe Amount (nmol) | Estimated Fold (log10) | uL Mix to add |
Hs-1 | U87MG | 1 | 100 nM | 0.01 | 6.8 | 2 |
Hs-2 | U87MG | 5 | 500 nM | 0.05 | 7.5 | 10 |
Hs-3 | U87MG | 10 | 1 uM | 0.1 | 7.8 | 20 |
Hs-4 | U87MG | 0 | 0 | 0 | NA | 0 |
mm-1 | 3T3 | 1 | 100 nM | 0.01 | 6.8 | 2 |
mm-2 | 3T3 | 5 | 500 nM | 0.05 | 7.5 | 10 |
mm-3 | 3T3 | 10 | 1 uM | 0.1 | 7.8 | 20 |
mm-4 | 3T3 | 0 | 0 | 0 | NA | 0 |
NTC | NA | 1 | 100 nM | 0.01 | NA | 2 |