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=C Probe Test 3-SOD1/VIM-Stringent Washes= [[Daniel:Notebook/ComboLock|Back to Calendar]] ==Protocol== ===Buffer Prep=== '''Stringent Wash Buffer''' {| class="wikitable" <hiddentext>generated with [[:de:Wikipedia:Helferlein/VBA-Macro for EXCEL tableconversion]] V1.8</hiddentext> |- style="background-color:#C4D79B;font-size:12pt;font-weight:bold" align="center" | width="110" height="42" | Reagent | width="65" | Stock | width="65" | Final | width="65" | Dilution | width="74" | Amt in 1 mL (uL) |- style="font-size:12pt" |style="font-weight:bold" height="15" valign="bottom" | PBS | align="center" valign="bottom" | 10X | align="center" valign="bottom" | 1X | align="center" align="center" valign="bottom" | 10 | align="center" valign="bottom" | 100 uL |- style="background-color:#D9D9D9;font-size:12pt" |style="font-weight:bold" height="15" valign="bottom" | SSC | align="center" valign="bottom" | 20X | align="center" valign="bottom" | 4X | align="center" align="center" valign="bottom" | 5 | align="center" valign="bottom" | 200 uL |- style="font-size:12pt" |style="font-weight:bold" height="15" valign="bottom" | Rnasin | align="center" valign="bottom" | 40000 U/mL | align="center" valign="bottom" | 40 U/mL | align="center" align="center" valign="bottom" | 1000 | align="center" valign="bottom" | 1 uL |- style="background-color:#D9D9D9;font-size:12pt" |style="font-weight:bold" height="15" valign="bottom" | nf H2O | align="center" valign="bottom" | NA | align="center" valign="bottom" | NA | align="center" valign="bottom" | NA | align="center" valign="bottom" | 700 uL |} '''LPH Buffer''' {| class="wikitable" <hiddentext>generated with [[:de:Wikipedia:Helferlein/VBA-Macro for EXCEL tableconversion]] V1.8</hiddentext> |- style="background-color:#C4D79B;font-size:12pt;font-weight:bold" align="center" | width="110" height="42" | Reagent | width="65" | Stock | width="65" | Final | width="65" | Dilution | width="90" | Amt in 1 mL (uL) |- style="font-size:12pt" |style="font-weight:bold" height="15" valign="bottom" | Oligos | align="center" valign="bottom" | 100 uM | align="center" valign="bottom" | 100 nM | align="center" align="center" valign="bottom" | 1000 | align="center" valign="bottom" | 1 uL |- style="background-color:#D9D9D9;font-size:12pt" |style="font-weight:bold" height="15" valign="bottom" | SSC | align="center" valign="bottom" | 20X | align="center" valign="bottom" | 1X | align="center" align="center" valign="bottom" | 20 | align="center" valign="bottom" | 50 uL |- style="font-size:12pt" |style="font-weight:bold" height="15" valign="bottom" | Rnasin | align="center" valign="bottom" | 40000 U/mL | align="center" valign="bottom" | 40 U/mL | align="center" align="center" valign="bottom" | 1000 | align="center" valign="bottom" | 1 uL |- style="background-color:#D9D9D9;font-size:12pt" |style="font-weight:bold" height="15" valign="bottom" | PBS | align="center" valign="bottom" | 10X | align="center" valign="bottom" | 1X | align="center" align="center" valign="bottom" | 10 | align="center" valign="bottom" | 100 uL |- style="font-size:12pt" |style="font-weight:bold" height="15" valign="bottom" | nf H2O | align="center" valign="bottom" | NA | align="center" valign="bottom" | NA | align="center" valign="bottom" | NA | align="center" valign="bottom" | 850 uL |} '''KLN Mix''' {| class="wikitable" <hiddentext>generated with [[:de:Wikipedia:Helferlein/VBA-Macro for EXCEL tableconversion]] V1.8</hiddentext> |- style="background-color:#C4D79B;font-size:12pt;font-weight:bold" align="center" | width="180" height="31" | Reagent | width="65" | Stock | width="65" | uL added |- style="font-size:12pt" | height="15" valign="bottom" | Amp Ligase Buffer | align="center" valign="bottom" | 10X | align="center" align="center" valign="bottom" | 2 |- style="background-color:#D9D9D9;font-size:12pt" | height="15" valign="bottom" | dNTPs | align="center" valign="bottom" | 100 uM | align="center" align="center" valign="bottom" | 2 |- style="font-size:12pt" | height="15" valign="bottom" | Hemo Klentaq | align="center" valign="bottom" | NA | align="center" align="center" valign="bottom" | 4 |- style="background-color:#D9D9D9;font-size:12pt" | height="15" valign="bottom" | Amp Ligase | align="center" valign="bottom" | 5 U/uL | align="center" align="center" valign="bottom" | 2 |- style="font-size:12pt" | height="15" valign="bottom" | nfH2O | align="center" valign="bottom" | NA | align="center" align="center" valign="bottom" | 10 |} Resuspended SOD1 C probes in 40 uL nfH2O each (100 uM) ===Experiment=== <ol> <li>C-Probe Hybridization</li> <ol type="A"> <li>Add cells to a new tube such that there are 100000 cells present</li> <ol type="a"><li>Human-500 uL; Mouse-140 uL</li></ol> <li>Pellet cells via centrifugation at 600g for 3 min</li> <li>Combine 1.5 uL of each C probe into a 0.2 mL tube</li> <li>Heat C probes to 90C for 5 min; Chill probes on ice to quench</li> <li>Add probes to CPH buffer for final concentration of 100 nM (Add 2 uL probe mixture to 0.5mL hybridization buffer)</li> {| class="wikitable" <hiddentext>generated with [[:de:Wikipedia:Helferlein/VBA-Macro for EXCEL tableconversion]] V1.8</hiddentext> |- style="font-size:12pt" align="center" | align="center" width="85" height="30" | |style="background-color:#CCC0DA;font-weight:bold" width="120" | C Probes-VIM (C1+C2) |style="background-color:#CCC0DA;font-weight:bold" width="120" | C Probes-SOD1 (C1+C2) |style="background-color:#CCC0DA;font-weight:bold" width="65" | U87MG |style="background-color:#CCC0DA;font-weight:bold" width="65" | 3T3 Cells |- style="font-size:12pt" | height="15" | Sample 1 | align="center" | X | align="center" | X | align="center" | X | align="center" align="center" | |- style="background-color:#D9D9D9;font-size:12pt" | height="15" | Sample 2 | align="center" align="center" | | align="center" align="center" | | align="center" | X | align="center" align="center" | |- style="font-size:12pt" | height="15" valign="bottom" | Sample 3 | align="center" | X | align="center" | X | align="center" align="center" | | align="center" | X |- style="background-color:#D9D9D9;font-size:12pt" | height="15" valign="bottom" | Sample 4 | align="center" align="center" | | align="center" align="center" | | align="center" align="center" | | align="center" | X |- style="font-size:12pt" | height="15" valign="bottom" | Sample 5 | align="center" valign="bottom" | X | align="center" valign="bottom" | X | align="center" align="center" valign="bottom" | | align="center" align="center" valign="bottom" | |} <li>Incubate probes at 40C for 1 hour with vigorous agitation</li> <li>Wash three times with 200 uL wash buffer and pelleting at 600xg for 3 min</li> <li>Incubate cells for 20 min at 40C in 200 uL stringent wash buffer</li> </ol> <li>Latch and Padlock Hybridization</li> <ol type="A"> <li>Pellet at 600xg for 3 min and remove supernatant</li> <li>Resuspend pellet in 100 uL LPH buffer with 100 nM Padlock0001-68 </li> <li>Incubate for 30 min at 37C</li> <li>Wash twice with 200 uL wash buffer by pelleting cells at 600xg for 3 min</li> <li>Incubate cells for 20 min at 40C in 200 uL stringent wash buffer</li> <li>Resuspend pellet in 100 uL LPH buffer with 100 nM Latch0001</li> <li>Incubate for 30 min at 37C</li> <li>Wash twice with 200 uL wash buffer by pelleting cells at 600xg for 3 min</li> <li>Incubate cells for 20 min at 40C in 200 uL stringent wash buffer</li> <li>Resuspend with 20 uL 1x Amp Ligase buffer</li></ol> <li>Circularization</li> <ol type="A"> <li>Prepare 20 uL KLN mix</li> <li>Add 2 uL KLN mix to 20 uL of reaction</li> <li>Incubate at 55C overnight (XX hours) </li> <li>Continued [[Daniel:Notebook/ComboLock/2016-7-19|tomorrow]]</li> </ol> [[Category:ComboLock]] [[Category:20160718]]
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