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=Cell Test with RCA= [[Daniel:Notebook/ComboLock|Back to Calendar]] This experiment is designed to see if I can get mRNA targeting C-Probes to work in cells, this time with RCA. I'll be trying both VIM and SOD1, just to have two separate genes. ==Buffer Prep== Need more C Probe Hybridization Buffer. I'll be freezing it down in aliquots of 500 uL. {| class="wikitable" <hiddentext>generated with [[:de:Wikipedia:Helferlein/VBA-Macro for EXCEL tableconversion]] V1.8</hiddentext> |- style="background-color:#6AFF6F;font-size:12pt;font-weight:bold" align="center" | width="180" height="30" | Reagent | width="85" | Stock | width="85" | Final | width="85" | Dilution | width="85" | Amount in 10 mL (uL) |- style="font-size:12pt" | height="15" valign="bottom" | SSC Buffer | align="center" valign="bottom" | 20X | align="center" valign="bottom" | 1X | align="center" align="center" valign="bottom" | 20 | align="center" align="center" valign="bottom" | 500 |- style="background-color:#BFBFBF;font-size:12pt" | height="15" valign="bottom" | Polyvinylsulonic Acid | align="center" align="center" valign="bottom" | 100% | align="center" align="center" valign="bottom" | 2.50% | align="center" align="center" valign="bottom" | 40 | align="center" align="center" valign="bottom" | 250 |- style="font-size:12pt" | height="15" valign="bottom" | Ribonucleoside Vanadyl Complex | align="center" valign="bottom" | 200 mM | align="center" valign="bottom" | 20 mM | align="center" align="center" valign="bottom" | 10 | align="center" align="center" valign="bottom" | 1000 |- style="background-color:#BFBFBF;font-size:12pt" | 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" align="center" valign="bottom" | 10 |- style="font-size:12pt" | height="15" valign="bottom" | 0.1% 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" align="center" valign="bottom" | 100 |- style="background-color:#BFBFBF;font-size:12pt" | height="15" valign="bottom" | Salmon Sperm DNA | align="center" valign="bottom" | 10 mg/mL | align="center" valign="bottom" | 100 ug/mL | align="center" align="center" valign="bottom" | 100 | align="center" align="center" valign="bottom" | 100 |- style="font-size:12pt" | height="15" valign="bottom" | DEPC Treated Water | align="center" valign="bottom" | NA | align="center" valign="bottom" | NA | align="center" valign="bottom" | NA | align="center" align="center" valign="bottom" | 8040 |} ==Protocol== '''Sample Matrix''' {| 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="140" height="45" | |style="background-color:#CCC0DA;font-weight:bold" width="105" | C Probes-VIM /SOD1 (C1v2+C2) |style="background-color:#CCC0DA;font-weight:bold" width="65" | U87MG |- style="font-size:12pt" | height="15" | Sample 1 (AB) | align="center" | X | align="center" | X |- style="background-color:#D9D9D9;font-size:12pt" | height="15" | Sample 2 (AB) | align="center" | X | align="center" | X |- style="font-size:12pt" | height="15" valign="bottom" | Sample 3 (AB) | align="center" align="center" | | align="center" | X |- style="background-color:#D9D9D9;font-size:12pt" | height="15" valign="bottom" | Sample 4 (AB) | align="center" | X | align="center" align="center" | |} <ol> <li>Cell Preparation</li> <ol type="A"> <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 2 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> <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 Padlock0201-68 and 100 nM Latch0001</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 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 8.2X uL Phusion mix</li> <li>Add 20 uL Phusion mix to 20 uL of reaction for sample 1</li> <li>Incubate at 55C for 2 hours</li> <li>Heat kill for 2 minutes at 94C and hold at 16C overnight; Continued [[Daniel:Notebook/ComboLock/2016-11-8|tomorrow]]</li> </ol> ===Buffers=== '''Phusion 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="150" height="30" | Reagent | width="85" | Stock Conc | width="85" | Final Amount | width="85" | 1x Vol (uL) | width="85" | MM Vol (8.2x) (uL) |- style="font-size:12pt" | height="15" valign="bottom" | NAD+ | align="center" valign="bottom" | 5 mM | align="center" valign="bottom" | 40 nmol | align="center" align="center" valign="bottom" | 8 | align="center" align="center" valign="bottom" | 65.6 |- style="background-color:#BFBFBF;font-size:12pt" | height="15" valign="bottom" | dNTP | align="center" valign="bottom" | 1 mM | align="center" valign="bottom" | 600 pmol | align="center" align="center" valign="bottom" | 0.6 | align="center" align="center" valign="bottom" | 4.92 |- style="font-size:12pt" | height="15" valign="bottom" | Betaine | align="center" valign="bottom" | 5 M | align="center" valign="bottom" | 15 umol | align="center" align="center" valign="bottom" | 3 | align="center" align="center" valign="bottom" | 24.6 |- style="background-color:#BFBFBF;font-size:12pt" | height="30" valign="bottom" | 10X AmpLigase Buffer | align="center" valign="bottom" | 10X | align="center" valign="bottom" | 1X | align="center" align="center" valign="bottom" | 2 | align="center" align="center" valign="bottom" | 16.4 |- style="font-size:12pt" | height="15" valign="bottom" | Amp Ligase | align="center" valign="bottom" | 5 U/uL | align="center" valign="bottom" | 10 U | align="center" align="center" valign="bottom" | 2 | align="center" align="center" valign="bottom" | 16.4 |- style="background-color:#BFBFBF;font-size:12pt" | height="30" valign="bottom" | Phusion HF DNA Polymerase | align="center" valign="bottom" | 2000 U/mL | align="center" valign="bottom" | 6.4U | align="center" align="center" valign="bottom" | 3.2 | align="center" align="center" valign="bottom" | 26.24 |- style="font-size:12pt" | height="15" valign="bottom" | nf H2O | align="center" align="center" valign="bottom" | | align="center" align="center" valign="bottom" | | align="center" align="center" valign="bottom" | 1.2 | align="center" align="center" valign="bottom" | 9.84 |- style="background-color:#BFBFBF;font-size:12pt" |style="font-weight:bold" height="15" valign="bottom" | Total | align="center" align="center" valign="bottom" | | align="center" align="center" valign="bottom" | | align="center" align="center" valign="bottom" | 20 | align="center" align="center" valign="bottom" | 164 |} '''ComboLock Wash Buffer''' {| class="wikitable" <hiddentext>generated with [[:de:Wikipedia:Helferlein/VBA-Macro for EXCEL tableconversion]] V1.8</hiddentext> |- style="background-color:#76933C;font-size:12pt;font-weight:bold" align="center" | width="82" height="30" | Reagent | width="56" | Stock | width="65" | Final | width="65" | Dilution | width="80" | Amount in 20 mL |- style="font-size:12pt" | 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" | 2 mL |- style="background-color:#BFBFBF;font-size:12pt" | 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" | 4 mL |- style="font-size:12pt" | 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" | 2 uL |- style="background-color:#BFBFBF;font-size:12pt" | height="15" valign="bottom" | nf H2O | valign="bottom" | NA | align="center" valign="bottom" | NA | valign="bottom" | NA | align="center" valign="bottom" | 14 mL |} [[Category:ComboLock]] [[Category:20161107]]
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