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=C Probe Hybridization Test= [[Daniel:Notebook/ComboLock|Back to Calendar]] 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=== <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]] <ol> <li>Fix cells (CERC)</li> <ol type="A"> <li>Start with confluent U87MG cells</li> <li>Remove media (EMEM) and wash with 1X PBS</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>Pellet remainder of the cells in a 15 mL Falcon tube at 600g for 3 min</li> <li>Remove supernatant</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 5 mL wash buffer</li> <ol type="a"> <li>add 0.5 uL RNasin (40 u/uL) to 5 mL wash buffer prime</li> </ol> <li>Pellet cells and add 5 mL ice-cold methanol to permeabilize; incubate 10 min on ice</li> </ol> </ol> ===Buffers=== <ol type="A"> <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>
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