Daniel:Notebook/ComboLock/2016-5-23: Difference between revisions
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>Djacobse |
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<ol type="I"> | <ol type="I"> | ||
<li>Wash Buffer</li> | <li>Wash Buffer</li> | ||
{| class="wikitable" <hiddentext>generated with [[:de:Wikipedia:Helferlein/VBA-Macro for EXCEL tableconversion]] V1.8</hiddentext> | |||
|- style="background-color:#B8CCE4;font-size:12pt;font-weight:bold" align="center" | |||
| width="101" height="30" | Reagent | |||
| width="85" | Stock Conc. | |||
| width="71" | Final Conc. | |||
| width="74" | Amt. Added | |||
|- style="font-size:12pt" | |||
| height="15" valign="bottom" | PBS | |||
| align="center" valign="bottom" | 10X | |||
| align="center" valign="bottom" | 1X | |||
| align="center" valign="bottom" | 5 mL | |||
|- style="background-color:#D9D9D9;font-size:12pt" | |||
| height="15" valign="bottom" | Tween 20 | |||
| align="center" align="center" valign="bottom" | 100% | |||
| align="center" align="center" valign="bottom" | 0.10% | |||
| align="center" valign="bottom" | 100 uL | |||
|- style="font-size:12pt" | |||
| height="15" valign="bottom" | Rnasin | |||
| align="center" valign="bottom" | 40000 U/mL | |||
| align="center" valign="bottom" | 4 U/mL | |||
| align="center" valign="bottom" | **5 uL | |||
|- style="background-color:#D9D9D9;font-size:12pt" | |||
| height="15" valign="bottom" | Water | |||
| align="center" valign="bottom" | NA | |||
| align="center" valign="bottom" | NA | |||
| align="center" valign="bottom" | 44.9 mL | |||
|- style="font-size:12pt" | |||
| height="15" valign="bottom" | Total Volume | |||
| align="center" align="center" valign="bottom" | | |||
| align="center" align="center" valign="bottom" | | |||
|style="font-weight:bold" align="center" valign="bottom" | 50 mL | |||
|} | |||
===Probe Hybridization=== | ===Probe Hybridization=== |
Revision as of 17:26, 23 May 2016
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
- Fix cells
- 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
- 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 |