Daniel:Notebook/ComboLock/2016-5-23: Difference between revisions
Jump to navigation
Jump to search
>Djacobse (Created page with "=C Probe Hybridization Test= Back to Calendar This will be the first test of the C Probe hybridization protocol for Combo-Lock. ==Protocol==...") |
>Djacobse |
||
Line 3: | Line 3: | ||
[[Daniel:Notebook/ComboLock|Back to Calendar]] | [[Daniel:Notebook/ComboLock|Back to Calendar]] | ||
This will be the first test of the C Probe hybridization protocol for Combo-Lock. | 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== | ==Protocol== | ||
Line 16: | Line 16: | ||
<li>Trypsinize cells using 0.5 mL trypsin LE express; incubate 5 min at 37C</li> | <li>Trypsinize cells using 0.5 mL trypsin LE express; incubate 5 min at 37C</li> | ||
<li>Resuspend cells in by adding 4.5 mL media; use 0.5 mL to passage</li> | <li>Resuspend cells in by adding 4.5 mL media; use 0.5 mL to passage</li> | ||
<li>Pellet remainder of the cells | <li>Pellet remainder of the cells in a 15 mL Falcon tube at 600g for 3 min</li> | ||
<li>Remove supernatant</li> | <li>Remove supernatant</li> | ||
<li>Fix cells in EMEM with 1.6% paraformaldehyde for 10 min at RT</li> | <li>Fix cells in 5 mL EMEM with 1.6% paraformaldehyde for 10 min at RT</li> | ||
<li>Pellet cells by centrifuging at 600xg for 3 min</li> | |||
<li>Wash cells with wash buffer</li> | <li>Wash cells with wash buffer</li> | ||
<li>Pellet cells and permeabilize with ice-cold methanol for 10 min on ice</li> | <li>Pellet cells and permeabilize with ice-cold methanol for 10 min on ice</li> | ||
Line 33: | Line 34: | ||
</ol> | </ol> | ||
</ol> | </ol> | ||
===Buffers=== | |||
<dl> | |||
<dt>Wash Buffer</dt> | |||
<dd>PBS</dd> | |||
<dd>0.1% Tween-20</dd> | |||
<dd>4 U/mL RNasin</dd> |
Revision as of 17:03, 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
- 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 wash buffer
- Pellet cells and permeabilize with ice-cold methanol for 10 min on ice Once in methanol cells may be stored long term
- 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
Buffers
- Wash Buffer
- PBS
- 0.1% Tween-20
- 4 U/mL RNasin