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=Phusion Test= [[Daniel:Notebook/ComboLock|Back to Calendar]] Hemo Klentaq has strand displacement activity, which could be a potential (big) problem for latch-padlock circularization. Therefore, on Chris and Andrew's recommendation I will try a test using Phusion instead of Hemo Klentaq. ==Buffers== '''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 |} '''Phusion Buffer''' {| 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="200" height="40" | Reagent | width="65" | Stock Conc | width="72" | Final Amount | width="65" | 1x Vol (uL) | width="88" | MM Vol (4.1x) (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" | 32.8 |- style="background-color:#D9D9D9;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" | 2.46 |- 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" | 12.3 |- style="background-color:#D9D9D9;font-size:12pt" | height="15" 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" | 8.2 |- 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" | 8.2 |- style="background-color:#D9D9D9;font-size:12pt" | height="15" 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" | 13.12 |- 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" | 4.92 |- style="background-color:#D9D9D9;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" | 82 |} ==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 (from [[Daniel:Notebook/ComboLock/2016-7-25|7-25-2016]])</li></ol> <li>Add cells to a new tube such that there are 30000 cells present per sample</li> <ol type="a"><li>Human- 350 uL (3 samples) uL; Mouse- 30 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 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> {| 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" | Klentaq |- 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" | Phusion |- style="font-size:12pt" | height="15" valign="bottom" | Sample 3 | align="center" align="center" | | align="center" align="center" | | align="center" | X | align="center" align="center" | | align="center" | Phusion |- style="background-color:#D9D9D9;font-size:12pt" | height="15" valign="bottom" | Sample 4 | align="center" | X | align="center" | X | align="center" align="center" | | align="center" | X | align="center" valign="bottom" | Phusion |- 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" | | align="center" | Phusion |} <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>Prepare 4.1x master mix of Phusion mix</li> <li>Add 20 uL Phusion mix to 20 uL of reaction for samples 2-5</li> <li>Add 2 uL KLN mix to 20 uL of reaction for sample 1</li> <li>Incubate at 55C overnight (XX hours) </li> <li>Continued [[Daniel:Notebook/ComboLock/2016-7-29|tomorrow]]</li> </ol> [[Category:ComboLock]] [[Category:20160728]]
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