Daniel:Notebook/ComboLock/2016-5-23: Difference between revisions
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==Protocol== | ==Protocol== | ||
===Buffer and Reagent Prep=== | |||
<ol type="I"> | |||
<li>Ribonucleoside Vanadyl Complex-to minimize freeze thaws, thaw at 65C for 10 min; store in 1 mL aliquots</li> | |||
<li>Resuspend following primers to 100 uM (pmol/uL)</li> | |||
<ol type="i"> | |||
<li>'''C Probes (40 uL to 100 uM)''': CAV1-C1/C2,GFAP-C1/C2,SOD1-C1/C2,VIM-C1/C2</li> | |||
<li>'''Primers (uL to 100 uM)''': P2 (295), P4_RC (295), P6 (316), P12-RC (323) </li> | |||
</ol> | |||
<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 | |||
|} | |||
<li>Hybridization Buffer-Make 10 mL at a time and store in 1 mL aliquots</li> | |||
{| class="wikitable" <hiddentext>generated with [[:de:Wikipedia:Helferlein/VBA-Macro for EXCEL tableconversion]] V1.8</hiddentext> | |||
|- style="background-color:#CCC0DA;font-size:12pt;font-weight:bold" align="center" | |||
| width="143" height="30" | Reagent | |||
| width="101" | Stock Conc. | |||
| width="86" | Final Conc. | |||
| width="108" | Amt. Added | |||
| width="65" | Amt Added | |||
|- style="font-size:12pt" | |||
| height="15" valign="bottom" | SSC | |||
| align="center" valign="bottom" | 20X | |||
| align="center" valign="bottom" | 1X | |||
| align="center" valign="bottom" | 50 uL | |||
| align="center" valign="bottom" | 500 uL | |||
|- 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" | 1 uL | |||
| align="center" valign="bottom" | 10 uL | |||
|- style="font-size:12pt" | |||
| height="15" valign="bottom" | Rnasin | |||
| align="center" valign="bottom" | 40000 U/mL | |||
| align="center" valign="bottom" | 40 U/mL | |||
| align="center" valign="bottom" | 1 uL | |||
| align="center" valign="bottom" | 10 uL | |||
|- style="background-color:#D9D9D9;font-size:12pt" | |||
| height="15" valign="bottom" | Riboside Vanadyl Complex | |||
| align="center" valign="bottom" | 200 mM | |||
| align="center" valign="bottom" | 20 mM | |||
| align="center" valign="bottom" | 100 uL | |||
| align="center" valign="bottom" | 1 mL | |||
|- style="font-size:12pt" | |||
| height="15" valign="bottom" | Poly Vinylsulfonic Acid | |||
| align="center" align="center" valign="bottom" | 25% | |||
| align="center" align="center" valign="bottom" | 2.50% | |||
| align="center" valign="bottom" | 100 uL | |||
| align="center" valign="bottom" | 1 mL | |||
|- style="background-color:#D9D9D9;font-size:12pt" | |||
| height="15" valign="bottom" | Salmon Sperm | |||
| align="center" valign="bottom" | 10 mg/mL | |||
| align="center" valign="bottom" | 100 ug/mL | |||
| align="center" valign="bottom" | 10 uL | |||
| align="center" valign="bottom" | 100 uL | |||
|- style="font-size:12pt" | |||
| height="15" valign="bottom" | Probes | |||
| align="center" valign="bottom" | 100 uM | |||
| align="center" valign="bottom" | 100 nM | |||
| align="center" valign="bottom" | 1 uL/probe | |||
| align="center" valign="bottom" | *** | |||
|- 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" | 740 uL | |||
| align="center" valign="bottom" | 7.4 mL | |||
|- style="font-size:12pt" | |||
| height="15" valign="bottom" | Total | |||
| align="center" valign="bottom" | | |||
| align="center" valign="bottom" | | |||
|style="font-weight:bold" align="center" valign="bottom" | 1 mL | |||
|style="font-weight:bold" align="center" valign="bottom" | 10 mL | |||
|} | |||
===Probe Hybridization=== | |||
[[Daniel:Protocols/CProbeLigate|Base Protocol]] | [[Daniel:Protocols/CProbeLigate|Base Protocol]] | ||
<ol> | <ol> | ||
<li>Fix cells</li> | <li>Fix cells (CERC)</li> | ||
<ol type="A"> | <ol type="A"> | ||
<li>Start with confluent U87MG cells</li> | <li>Start with confluent U87MG cells</li> | ||
Line 20: | Line 144: | ||
<li>Fix cells in 5 mL 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>Pellet cells by centrifuging at 600xg for 3 min</li> | ||
<li>Wash cells with wash buffer</li> | <li>Wash cells with 5 mL wash buffer</li> | ||
<li> | <ol type="a"> | ||
<li>add 0.5 uL RNasin (40 u/uL) to 5 mL wash buffer prime</li> | |||
</ol> | </ol> | ||
<li>Pellet cells and add 5 mL ice-cold methanol to permeabilize; incubate 10 min on ice</li> | |||
<li>Pellet cells | |||
</ol> | </ol> | ||
</ol> | </ol> | ||
Line 38: | Line 155: | ||
<ol type="A"> | <ol type="A"> | ||
<li> '''Wash Buffer''': 1X PBS, 0.1% Tween-20, 4 U/mL RNasin</li> | <li>'''Wash Buffer''': 1X PBS, 0.1% Tween-20, 4 U/mL RNasin</li> | ||
<li>'''Hybridization Buffer''': 1X SSC, 20mM RVC, 40 U/mL RNasin, 0.1% Tween 20, 100 ug/mL salmon sperm DNA</li> | |||
</ol> | </ol> |
Latest revision as of 20:01, 23 May 2016
C Probe Hybridization Test[edit]
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[edit]
Buffer and Reagent Prep[edit]
- Ribonucleoside Vanadyl Complex-to minimize freeze thaws, thaw at 65C for 10 min; store in 1 mL aliquots
- Resuspend following primers to 100 uM (pmol/uL)
- C Probes (40 uL to 100 uM): CAV1-C1/C2,GFAP-C1/C2,SOD1-C1/C2,VIM-C1/C2
- Primers (uL to 100 uM): P2 (295), P4_RC (295), P6 (316), P12-RC (323)
- 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
- Wash Buffer: 1X PBS, 0.1% Tween-20, 4 U/mL RNasin
- Hybridization Buffer: 1X SSC, 20mM RVC, 40 U/mL RNasin, 0.1% Tween 20, 100 ug/mL salmon sperm DNA
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 |