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=BSA Ab-Oligo Linkage (From [[Daniel:Notebook/ComboLock/2016-10-14|Friday]])= [[Daniel:Notebook/ComboLock|Back to Calendar]] Friday I accidentally let the post-sulfo SMCC reaction elute into the wash container, which probably messed things up. So I'm repeating that part of the experiment and will mix the new Ab fraction with the oligo fraction prepared on [[Daniel:Notebook/ComboLock/2016-10-14|Friday]]. ==Protocol== <ol> <li>Buffer Prep</li> <ol type="A"><li>Mix 1.4 mL of monobasic and 3.6 mL of dibasic and 45 mL ddH2O to make 50 mL of 100 mM (buffer pH 7.2)</li></ol> <li>Antibody Activation</li> <ol type="A"> <li>Resuspend antibodies at 1 mg/mL in PBS</li> <li>Equilibrate a 40 kDa spin column (or plate) 4 times with 100 mM phosphate buffer, pH 7.3; 250 uL, spin at 1000xg for 2 min</li> <li>Add 20 uL antibody to the column and spin for 3 min at 1000xg; collect in a new plate</li> <li>Dissolve sulfo-SMCC to 3.33 mM in 100mM phosphate buffer</li> <li>Add 2 uL sulfo-SMCC to sample and 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> <li>Oligonucleotide Activation</li> <li>Ab-Oligo Mixing</li> <ol type="A"> <li>Mix SMCC-treated antibodies with DTT-treated oligonucleotides at 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> <li>Store at 4C</li> </ol></ol> =Padlock0301 Dilutions Test (Started [[Daniel:Notebook/ComboLock/2016-10-13|Thursday 10-13]])= I'm going to repeat the PCR test, just in case. <ol start="8"> <li>qPCR</li> <ol type="A"> <li>Prepare the following master mix</li> <ol type="a"> <li>334.25 uL nfH2O</li> <li>9.55 uL 100 uM AmpF-CLv2</li> <li>477.5 uL Kapa SYBR Fast MM</li></ol> <li>Add 43 uL master mix to each well</li> <li>Add sample and AmpR indexes according to sample table in the next section</li> <li>qPCR Cycles</li> <ol type="a"> <li>95C 3 min</li> <li>95C 3 sec</li> <li>55C 30 sec</li> <li>72C 20 sec</li> <li>plate read</li> <li>goto b x24</li> <li>72C 2 min</li> <li>16C hold</li> </ol></ol> <li>TBE Gel</li> <ol type="A"> <li>Mix 176 uL TBE, 44 uL 6x loading dye</li> <li>Aliquot 10 uL per sample/ladder lane onto parafilm</li> <li>Add 2 uL of sample or ladder to correct drop</li> <li>Load 10 uL in to well</li> <li>Run gel for 22 minutes at 250V</li> <li>Open gel and stain with 3 uL SYBR Gold for 3 minutes</li> <li>Rinse gel and image in gel doc</li> </ol></ol> ==qPCR Sample Table== {| class="wikitable" <hiddentext>generated with [[:de:Wikipedia:Helferlein/VBA-Macro for EXCEL tableconversion]] V1.8</hiddentext> |- style="background-color:#92CDDC;font-size:12pt;font-weight:bold" align="center" | width="109" height="30" | Sample | width="97" | Template Molecule Count | width="65" | Lane | width="65" | AmpR Index | width="65" | Sample Vol (uL) | width="79" | 2X Kapa SYBR qPCR MM | width="65" | 100 uM AmpF-CLv2 | width="89" | 10 uM AmpR6.3-IndXX | width="65" | H2O | width="77" | Total Volume (uL) |- style="font-size:12pt" align="center" valign="bottom" |style="font-weight:bold" height="15" | Sample 6A | 10 amol | A1 | align="center" | 20 | align="center" | 2 | align="center" | 25 | align="center" | 0.5 | align="center" | 5 | align="center" | 17.5 | align="center" | 50 |- style="background-color:#BFBFBF;font-size:12pt" align="center" valign="bottom" |style="font-weight:bold" height="15" | Sample 6B | 10 amol | B1 | align="center" | 20 | align="center" | 2 | align="center" | 25 | align="center" | 0.5 | align="center" | 5 | align="center" | 17.5 | align="center" | 50 |- style="font-size:12pt" align="center" valign="bottom" |style="font-weight:bold" height="15" | Sample 6C | 10 amol | C1 | align="center" | 20 | align="center" | 2 | align="center" | 25 | align="center" | 0.5 | align="center" | 5 | align="center" | 17.5 | align="center" | 50 |- style="background-color:#BFBFBF;font-size:12pt" align="center" valign="bottom" |style="font-weight:bold" height="15" | Sample 8A | 100 zmol | D1 | align="center" | 21 | align="center" | 2 | align="center" | 25 | align="center" | 0.5 | align="center" | 5 | align="center" | 17.5 | align="center" | 50 |- style="font-size:12pt" align="center" valign="bottom" |style="font-weight:bold" height="15" | Sample 8B | 100 zmol | E1 | align="center" | 21 | align="center" | 2 | align="center" | 25 | align="center" | 0.5 | align="center" | 5 | align="center" | 17.5 | align="center" | 50 |- style="background-color:#BFBFBF;font-size:12pt" align="center" valign="bottom" |style="font-weight:bold" height="15" | Sample 8C | 100 zmol | F1 | align="center" | 21 | align="center" | 2 | align="center" | 25 | align="center" | 0.5 | align="center" | 5 | align="center" | 17.5 | align="center" | 50 |- style="font-size:12pt" align="center" valign="bottom" |style="font-weight:bold" height="15" | Sample 9A | 10 zmol | G1 | align="center" | 22 | align="center" | 2 | align="center" | 25 | align="center" | 0.5 | align="center" | 5 | align="center" | 17.5 | align="center" | 50 |- style="background-color:#BFBFBF;font-size:12pt" align="center" valign="bottom" |style="font-weight:bold" height="15" | Sample 9B | 10 zmol | H1 | align="center" | 22 | align="center" | 2 | align="center" | 25 | align="center" | 0.5 | align="center" | 5 | align="center" | 17.5 | align="center" | 50 |- style="font-size:12pt" align="center" valign="bottom" |style="font-weight:bold" height="15" | Sample 9C | 10 zmol | A12 | align="center" | 22 | align="center" | 2 | align="center" | 25 | align="center" | 0.5 | align="center" | 5 | align="center" | 17.5 | align="center" | 50 |- style="background-color:#BFBFBF;font-size:12pt" align="center" valign="bottom" |style="font-weight:bold" height="15" | Sample 10A | 1 zmol | B12 | align="center" | 23 | align="center" | 2 | align="center" | 25 | align="center" | 0.5 | align="center" | 5 | align="center" | 17.5 | align="center" | 50 |- style="font-size:12pt" align="center" valign="bottom" |style="font-weight:bold" height="15" | Sample 10B | 1 zmol | C12 | align="center" | 23 | align="center" | 2 | align="center" | 25 | align="center" | 0.5 | align="center" | 5 | align="center" | 17.5 | align="center" | 50 |- style="background-color:#BFBFBF;font-size:12pt" align="center" valign="bottom" |style="font-weight:bold" height="15" | Sample 10C | 1 zmol | D12 | align="center" | 23 | align="center" | 2 | align="center" | 25 | align="center" | 0.5 | align="center" | 5 | align="center" | 17.5 | align="center" | 50 |- style="font-size:12pt" align="center" valign="bottom" |style="font-weight:bold" height="15" | Sample 11A | 100 ymol | E12 | align="center" | 24 | align="center" | 2 | align="center" | 25 | align="center" | 0.5 | align="center" | 5 | align="center" | 17.5 | align="center" | 50 |- style="background-color:#BFBFBF;font-size:12pt" align="center" valign="bottom" |style="font-weight:bold" height="15" | Sample 11B | 100 ymol | F12 | align="center" | 24 | align="center" | 2 | align="center" | 25 | align="center" | 0.5 | align="center" | 5 | align="center" | 17.5 | align="center" | 50 |- style="font-size:12pt" align="center" valign="bottom" |style="font-weight:bold" height="15" | Sample 11C | 100 ymol | G12 | align="center" | 24 | align="center" | 2 | align="center" | 25 | align="center" | 0.5 | align="center" | 5 | align="center" | 17.5 | align="center" | 50 |- style="background-color:#BFBFBF;font-size:12pt" align="center" valign="bottom" |style="font-weight:bold" height="15" | Sample 12A | 10 ymol | H12 | align="center" | 25 | align="center" | 2 | align="center" | 25 | align="center" | 0.5 | align="center" | 5 | align="center" | 17.5 | align="center" | 50 |- style="font-size:12pt" align="center" valign="bottom" |style="font-weight:bold" height="15" | Sample 12B | 10 ymol | A2 | align="center" | 25 | align="center" | 2 | align="center" | 25 | align="center" | 0.5 | align="center" | 5 | align="center" | 17.5 | align="center" | 50 |- style="background-color:#BFBFBF;font-size:12pt" align="center" valign="bottom" |style="font-weight:bold" height="15" | Sample 12C | 10 ymol | B2 | align="center" | 25 | align="center" | 2 | align="center" | 25 | align="center" | 0.5 | align="center" | 5 | align="center" | 17.5 | align="center" | 50 |- style="font-size:12pt" align="center" valign="bottom" |style="font-weight:bold" height="15" | NTC | align="center" | 0 | C2 | align="center" | 26 | align="center" | 2 | align="center" | 25 | align="center" | 0.5 | align="center" | 5 | align="center" | 19.5 | align="center" | 50 |} ==Results== <gallery perrow=2 heights=300px widths=300px> File:20161017-qPCR-Padlock0301-Dilutions2.png|qPCR image File:2016-10-17-Padlock0301-Dilutions2-Gel1.png|Gel image 1 File:2016-10-17-Padlock0301-Dilutions2-Gel2.png|Gel image 2 </gallery> =Cell Trial= <ol> <li>C-Probe Hybridization</li> <ol type="A"> <li>Measure cell concentrations in cell counter</li> <li>Add cells to a new tube such that there are 100,000 cells present 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="125" | C Probes-VIM (C1v2+C2) |style="background-color:#CCC0DA;font-weight:bold" width="65" | U87MG |- style="font-size:12pt" | height="15" | Sample 1 | align="center" | X | align="center" | X |- style="background-color:#D9D9D9;font-size:12pt" | height="15" | Sample 2 | align="center" | X | align="center" | X |- style="font-size:12pt" | height="15" valign="bottom" | Sample 3 | align="center" align="center" | | align="center" | X |- style="background-color:#D9D9D9;font-size:12pt" | height="15" valign="bottom" | Sample 4 | align="center" | X | align="center" align="center" | |} <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 20 uL KLN mix</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/2016-10-18|tomorrow]]</li> </ol> [[Category:ComboLock]] [[Category:20161017]]
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