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=Sequential Hybridization Test= [[Daniel:Notebook/ComboLock|Back to Calendar]] Today I'm going to try testing the latch and padlock hybridizations sequentially. This is because of the unusual results of the C probe tests from and [[Daniel:Notebook/ComboLock/2016-6-30|June 30]] and [[Daniel:Notebook/ComboLock/2016-7-2|July 2]]. ==Protocol== ===Buffer Prep=== '''C Probe Hyridization (CPH) 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="175" height="30" | Reagent | width="65" | Stock Conc. | width="65" | Final Conc. | width="65" | 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="30" 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="30" 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" align="center" valign="bottom" | | align="center" align="center" valign="bottom" | |style="font-weight:bold" align="center" valign="bottom" | 1 mL |style="font-weight:bold" align="center" valign="bottom" | 10 mL |} '''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 |} ===Experiment=== <ol> <li>C-Probe Hybridization</li> <ol type="A"> <li>Transfer cells to a new tube such that there are 100000 cells present</li> <ol type="a"><li>Human-1000 uL; Mouse-85 uL</li> <li>Human cells from [[Daniel:Notebook/ComboLock/2016-7-2#Human Cell Line Preparation|July 2]]; 1.17E5 cells/mL</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 Latch0001</li> <li>Incubate for 30 min at 37C</li> <li>Wash twice with 200 uL wash buffer</li> <ol type="a"><li>Wash by pelleting cells at 600xg for 3 min</li></ol> <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</li> <ol type="a"><li>Wash by pelleting cells at 600xg for 3 min</li></ol> <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/2016-7-6|tomorrow]]</li> </ol> =SOD1 Antibody Prep= Since I have the VIM antibody and C Probes ready and only the SOD1 C Probe, it makes sense to have this antibody as well. ==Protocol== <ol> <li>Prep</li> <ol type="A"> <li>Resuspend SOD1-PA in 63 uL nfH2O (1 mM) </li> <li>Resuspend SOD1-PB in 47 uL nfH2O (1 mM)</li> </ol> <li>Antibody Activation</li> <ol type="A"> <li>Resuspend SOD1 with 100 uL PBS (final conc 1 mg/mL)</li> <li>Do this twice:</li> <ol type="a"> <li>Equilibrate a 40 kDa spin column 4 times with 100 uL PBS, (100 mM pH 7.3) <ol type="i"><li>Spin at 1000xg for 2 min</li></ol> <li>Add 20 uL antibody to the column and spin for 3 min at 1000xg; collect in a new tube</li> <li>Add 2 uL 3.3 mM sulfo-SMCC in 100 mM phosphate buffer to sample</li> <ol type="i"><li>Stock is 100 mM sulfo-SMCC in 100 mM phosphate buffer</li> <li>Mix 3.3 uL stock with 96 uL 100 mM phosphate buffer (dilute from 1 M stock)</li></ol> <li>Incubate for 2 hr at 4C with 3 times intermittent mixing</li> <li>Equilibrate a new 40 kDa column with 100 mM phosphate buffer with 20 mM EDTA</li> <li>Transfer antibodies to new column and spin for 3 min at 1000xg</li> </ol> </ol> <li>Oligonucleotide Activation</li> <ol type="A"> <li>For VIM-PA and VIM-PB:</li> <ol type="a"> <li>Add 1.3 uL 100 uM oligonucleotide to tube</li> <li>Add 2.2 uL 40 mM DTT</li> <li>Incubate at 95C for 2 min followed by 1 hr at 37C</li> <li>Add 20 uL PBS with 20 mM EDTA</li> <li>Remove excess DTT using two consecutive [[Media:Zeba_Spin_Desalt_Colomn_7K_MWCO_UG_Manual.pdf|Zeba 7 kDa]] columns equilibrated with 100 mM PBS (10X PBS)</li> <ol type="a"><li>Spin at 1500xg for 1 minute for equilibrium; 2 min for sample</li></ol> </ol> </ol> <li>Ab-Oligo Mixing</li> <ol type="A"><li>For VIM-PA and VIM-PB:</li> <ol type="a"> <li>Mix SMCC-treated antibodies (22 uL) with DTT-treated oligonucleotides (35 uL)</li> <ol type="i"><li>Amounts to 10X molar excess of oligo to Ab</li> <li>Transfer to pre-wet [[Media:SlideALyzer_MINI_Dialy_Unit_UG_Manual.pdf|Slide-A-Lyzer Mini 7 MWCO dialysis cup]]</li> <li>Dialyze in 1 L PBS with 5 mM EDTA at 4C for 2 days with one buffer exchange to PBS</li> <li>Dilute probes to 75 ug/mL in PBS buffer with 4 mM EDTA, 35 ug/mL ssDNA, 0.1% fish gelatin, and 20 mM Tris HCl</li> </ol> [[Category:ComboLock]] [[Category:20160705]]
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