Daniel:Notebook/ComboLock/2016-5-23
Jump to navigation
Jump to search
C Probe Hybridization Test
This will be the first test of the C Probe hybridization protocol for Combo-Lock. I have been growing U87MG cells for several days now and am ready to passage them. When I passage I'll take the remaining 90% for this test. Since I don't have the antibodies ready yet (I am waiting on some columns) I am just testing the C Probes.
Protocol
Buffer and Reagent Prep
- Wash Buffer
- Hybridization Buffer-Make 10 mL at a time and store in 1 mL aliquots
- Fix cells (CERC)
- Start with confluent U87MG cells
- Remove media (EMEM) and wash with 1X PBS
- Trypsinize cells using 0.5 mL trypsin LE express; incubate 5 min at 37C
- Resuspend cells in by adding 4.5 mL media; use 0.5 mL to passage
- Pellet remainder of the cells in a 15 mL Falcon tube at 600g for 3 min
- Remove supernatant
- Fix cells in 5 mL EMEM with 1.6% paraformaldehyde for 10 min at RT
- Pellet cells by centrifuging at 600xg for 3 min
- Wash cells with 5 mL wash buffer
- add 0.5 uL RNasin (40 u/uL) to 5 mL wash buffer prime
- Pellet cells and add 5 mL ice-cold methanol to permeabilize; incubate 10 min on ice
- Hybridization (Lab)
- Pellet cells via centrifugation at 600g for 3 min
- Heat C probes to 90C for 5 min; Chill probes on ice to quench
- Add probes to cells for final concentration of 100 nM
- 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
- Wash Buffer: 1X PBS, 0.1% Tween-20, 4 U/mL RNasin
Reagent | Stock Conc. | Final Conc. | Amt. Added |
PBS | 10X | 1X | 5 mL |
Tween 20 | 100% | 0.10% | 100 uL |
Rnasin | 40000 U/mL | 4 U/mL | 5 uL |
Water | NA | NA | 44.9 mL |
Total Volume | 50 mL |
Reagent | Stock Conc. | Final Conc. | Amt. Added | Amt Added |
SSC | 20X | 1X | 50 uL | 500 uL |
Tween 20 | 100% | 0.10% | 1 uL | 10 uL |
Rnasin | 40000 U/mL | 40 U/mL | 1 uL | 10 uL |
Riboside Vanadyl Complex | 200 mM | 20 mM | 100 uL | 1 mL |
Poly Vinylsulfonic Acid | 25% | 2.50% | 100 uL | 1 mL |
Salmon Sperm | 10 mg/mL | 100 ug/mL | 10 uL | 100 uL |
Probes | 100 uM | 100 nM | 1 uL/probe | *** |
Water | NA | NA | 740 uL | 7.4 mL |
Total | 1 mL | 10 mL |