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=Latch 100 and Dextran Test= [[Daniel:Notebook/ComboLock|Back to Calendar]] ==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="65" height="30" | Reagent | width="65" | Stock | width="65" | Final | width="65" | Dilution | width="65" | Amt in 5 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" | 500 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" | 1000 uL |- style="font-size:12pt" |style="font-weight:bold" height="30" 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" | 5 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" | 4.5 mL |} '''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="65" height="30" | Reagent | width="105" | Stock | width="65" | Final | width="65" | Dilution | width="65" | 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" | 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" | 1X | align="center" align="center" valign="bottom" | 20 | align="center" valign="bottom" | 50 uL |- style="font-size:12pt" |style="font-weight:bold" height="30" 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="30" 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 |} '''CPH Buffer with 10% Dextran Sulfate''' {| class="wikitable" <hiddentext>generated with [[:de:Wikipedia:Helferlein/VBA-Macro for EXCEL tableconversion]] V1.8</hiddentext> |- style="background-color:#D8E4BC;font-size:12pt;font-weight:bold" align="center" | width="165" height="30" | Reagent | width="105" | Stock Conc. | width="105" | Final Conc. | width="105" | 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 |- style="background-color:#D9D9D9;font-size:12pt" | height="15" valign="bottom" | Tween 20 | align="center" align="center" valign="bottom" | 10% | align="center" align="center" valign="bottom" | 0.10% | align="center" valign="bottom" | 10 uL |- style="font-size:12pt" | height="21" valign="bottom" | Rnasin | align="center" valign="bottom" | 40000 U/mL | align="center" valign="bottom" | 40 U/mL | align="center" valign="bottom" | 1 uL |- style="background-color:#D9D9D9;font-size:12pt" | height="30" valign="bottom" | Riboside Vanadyl Complex | align="center" valign="bottom" | 200 mM | align="center" valign="bottom" | 20 mM | align="center" valign="bottom" | 100 uL |- 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 |- 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 |- style="font-size:12pt" | height="15" valign="bottom" | Dextran Sulfate | align="center" align="center" valign="bottom" | 25% | align="center" align="center" valign="bottom" | 10% | align="center" valign="bottom" | 400 uL |- style="background-color:#D9D9D9;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 |- style="font-size:12pt" | height="15" valign="bottom" | Water | align="center" valign="bottom" | NA | align="center" valign="bottom" | NA | align="center" valign="bottom" | 340 uL |- style="background-color:#D9D9D9;font-size:12pt" | height="15" valign="bottom" | Total | align="center" align="center" valign="bottom" | | align="center" align="center" valign="bottom" | |style="font-weight:bold" align="center" valign="bottom" | 1 mL |} '''Wash Buffer Prime''' (Wash buffer with 2.5% polyvinyl sulfonic acid since it is better at removing free pigment from hematoxylin) {| 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="185" height="30" | Reagent | width="65" | Stock | width="65" | Final | width="65" | Dilution | width="65" | Amt in 10 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" | 1000 uL |- style="background-color:#D9D9D9;font-size:12pt" |style="font-weight:bold" height="30" valign="bottom" | Poly Vinylsulfonic Acid | align="center" valign="bottom" | 25% w/v | align="center" valign="bottom" | 2.5% w/v | align="center" align="center" valign="bottom" | 10 | align="center" valign="bottom" | 1000 uL |- style="font-size:12pt" |style="font-weight:bold" height="15" valign="bottom" | Tween 20 | align="center" align="center" valign="bottom" | 10% | align="center" align="center" valign="bottom" | 0.10% | align="center" align="center" valign="bottom" | 100 | align="center" valign="bottom" | 100 uL |- style="background-color:#D9D9D9;font-size:12pt" |style="font-weight:bold" height="30" valign="bottom" | Rnasin | align="center" valign="bottom" | 40000 U/mL | align="center" valign="bottom" | 4 U/mL | align="center" align="center" valign="bottom" | 10000 | align="center" valign="bottom" | 1 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" | 7.9 mL |} ==Protocol== <ol> <li>C-Probe Hybridization</li> <ol type="A"> <li>Measure cell concentrations in cell counter</li> <ol type="a"><li>Human - 3.05E5 cells/mL; Mouse - 9.75E5 cells/mL</li></ol> <li>Add cells to a new tube such that there are 25000 cells present per sample</li> <ol type="a"><li>Human- 328 uL (4 samples) uL; Mouse- 26 uL</li></ol> <li>Pellet cells via centrifugation at 600g for 3 min</li> <li>Wash with 200 uL wash buffer and pellet cells via centrifugation at 600g for 3 min</li> <li>Add 100 uL 50% hematoxylin (filtered) and pellet cells via centrifugation at 600g for 3 min</li> <li>Wash once with 200 uL wash buffer and pellet cells via centrifugation at 600g for 3 min</li> <li>Wash 2 times with 200 uL wash buffer prime and pellet cells via centrifugation at 600g for 3 min</li> <li>Pellet cells via centrifugation at 600g for 3 min</li> <li>Combine 0.5 uL of each C probe into a 0.2 mL tube with 3 uL nfH2O</li> <li>Heat C probes to 90C for 5 min; Chill probes on ice to quench</li> <li>Add probes to CPH buffer according to sample matrix, 1 uL probe mix per sample</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="105" height="45" | |style="background-color:#CCC0DA;font-weight:bold" width="65" | C Probes-VIM (C1+C2) |style="background-color:#CCC0DA;font-weight:bold" width="65" | 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="background-color:#CCC0DA;font-weight:bold" width="65" | Treatment |- style="font-size:12pt" | height="15" | Sample 1 | align="center" | X | align="center" | X | align="center" | X | align="center" align="center" | | align="center" | None |- style="background-color:#D9D9D9;font-size:12pt" | height="15" | Sample 2 | align="center" | X | align="center" | X | align="center" | X | align="center" align="center" | | align="center" | Dextran |- style="font-size:12pt" | height="15" valign="bottom" | Sample 3 | align="center" | X | align="center" | X | align="center" | X | align="center" align="center" | | align="center" | 10X Latch |- style="background-color:#D9D9D9;font-size:12pt" | height="15" valign="bottom" | Sample 4 | align="center" align="center" | | align="center" align="center" | | align="center" | X | align="center" align="center" | | align="center" valign="bottom" | None |- style="font-size:12pt" | height="15" valign="bottom" | Sample 5 | align="center" | X | align="center" | X | align="center" align="center" | | align="center" | X | align="center" | None |- style="background-color:#D9D9D9;font-size:12pt" | height="15" valign="bottom" | Sample 6 | align="center" valign="bottom" | X | align="center" valign="bottom" | X | align="center" align="center" valign="bottom" | | align="center" align="center" valign="bottom" | | align="center" | None |} <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 100 uL wash buffer by pelleting cells at 600xg for 3 min</li> <li>Incubate cells for 20 min at 40C in 150 uL stringent wash buffer</li> <li>Resuspend pellet in 100 uL LPH buffer with 100 nM Latch0001</li> <ol type="a"><li>For sample 3, use 10 uL 100 uM Latch0001 (10 uM final concentration)</li></ol> <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 and transfer to a 0.2 mL tube</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/2016-7-26|tomorrow]]</li> </ol> [[Category:ComboLock]] [[Category:20160725]]
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