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=Antibody Detection Test-Positive Control= [[Daniel:Notebook/ComboLock|Back to Calendar]] This will be a basic test of the antibody detection. ==Protocol== '''Sample Matrix''' {| class="wikitable" <hiddentext>generated with [[:de:Wikipedia:Helferlein/VBA-Macro for EXCEL tableconversion]] V1.8</hiddentext> |- style="background-color:#CCC0DA;font-size:12pt;font-weight:bold" align="center" | width="180" height="32" | Sample | width="200" | Condition |- style="font-size:12pt" | height="15" valign="bottom" | Sample 1 (AB) | align="center" valign="bottom" | Normal |- style="background-color:#BFBFBF;font-size:12pt" | height="15" valign="bottom" | Sample 2 (AB) | align="center" valign="bottom" | No Template |- style="font-size:12pt" | height="15" valign="bottom" | Sample 3 (AB) | align="center" valign="bottom" | No Antibodies |} <ol> <li>Template-Bead Binding</li> <ol type="A"> <li>Suspend 2.5 uL (25 ug) beads per sample in 100 uL wash buffer </li> <li>Apply magnet for 30 sec and remove supernatant</li> <li>Prepare following mixes <ol type="a"> <li>4X mix (samples 1,3): 80 uL Dynabuffer, 72 uL nfH<sub>2</sub>O, 8 uL BSA-biotin 0.2 mg/mL (6 pmol, 6X excess)</li> <li>2X mix (sample 2): 40 uL Dynabuffer, 36 uL nfH<sub>2</sub>O, 4 uL BSA-biotin 0.2 mg/mL (6 pmol, 6X excess)</li></ol> <li>Add 40 uL per sample and incubate at RT for 15 min</li> <li>Wash beads with 100 uL wash buffer; vortex to suspend; apply magnet and remove supernatant</li> </ol> ****** <li>Antibody hybridization</li> <ol type="A"> <li>Combine following into 0.2 mL tubes</li> {| class="wikitable" <hiddentext>generated with [[:de:Wikipedia:Helferlein/VBA-Macro for EXCEL tableconversion]] V1.8</hiddentext> |- style="background-color:#B1A0C7;font-size:12pt;font-weight:bold" align="center" | width="180" height="30" | Reagent | width="95" | uL per Sample | width="100" | Exp uL Total (4X 1,2) | width="100" | No Ab uL Total (2X-Sample 3) |- style="font-size:12pt" | height="15" valign="bottom" | Ab-A (75 ug/mL-0.5 uM) | align="center" align="center" | 2 | align="center" align="center" | 8 | align="center" align="center" valign="bottom" | 0 |- style="background-color:#BFBFBF;font-size:12pt" | height="15" valign="bottom" | Ab-B (75 ug/mL-0.5 uM) | align="center" align="center" | 2 | align="center" align="center" | 8 | align="center" align="center" valign="bottom" | 0 |- style="font-size:12pt" | height="15" valign="bottom" | PBS+EDTA (2X+10 mM) | align="center" align="center" | 10 | align="center" align="center" | 40 | align="center" align="center" valign="bottom" | 20 |- style="background-color:#BFBFBF;font-size:12pt" | height="15" valign="bottom" | nfH2O | align="center" align="center" | 6 | align="center" align="center" | 24 | align="center" align="center" valign="bottom" | 20 |- style="font-size:12pt;font-weight:bold" | height="15" valign="bottom" | Total | align="center" align="center" | 20 | align="center" align="center" | 80 | align="center" align="center" valign="bottom" | 20 |} <li>Add probes to cell sample for final concentration of 0.5 nM</li> <li>Incubate 1.5 hours at RT</li> <li>Wash beads with 100 uL wash buffer with 0.05% Tween 20; apply magnet and remove supernatant</li> <li>Repeat wash step above</li> <li>Repeat wash step above</li> </ol> </ol> ==Protocol-Part 2== <ol start="3"> <li>Latch and Bolt Hybridization/Ligation</li> <ol type="A"> <li>Set up the following master mix</li> {| 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="180" height="30" | Reagent | width="95" | Stock Conc | width="95" | Final Conc./Amt | width="95" | uL added | width="95" | Master Mix (6.2X) |- style="font-size:12pt" | height="15" valign="bottom" | T4 Ligase Reaction Buffer | align="center" | 5X | align="center" | 1X | align="center" align="center" | 4 | align="center" align="center" valign="bottom" | 24.8 |- style="background-color:#BFBFBF;font-size:12pt" | height="15" valign="bottom" | Latch Oligo | align="center" | 10 uM | align="center" | 10 pmol | align="center" align="center" | 1 | align="center" align="center" valign="bottom" | 6.2 |- style="font-size:12pt" | height="15" valign="bottom" | Bolt Oligo | align="center" | 10 uM | align="center" | 10 pmol | align="center" align="center" | 1 | align="center" align="center" valign="bottom" | 6.2 |- style="background-color:#BFBFBF;font-size:12pt" | height="15" valign="bottom" | Dephosphorylated Bolt | align="center" | 2.5 uM | align="center" | 10 pmol | align="center" align="center" | 8 | align="center" align="center" valign="bottom" | 0 |- style="font-size:12pt" | height="15" valign="bottom" | nfH2O | align="center" | NA | align="center" | NA | align="center" align="center" | 13 | align="center" align="center" valign="bottom" | 80.6 |- style="background-color:#BFBFBF;font-size:12pt" |style="font-weight:bold" height="15" valign="bottom" | Total | align="center" align="center" | | align="center" align="center" | |style="font-weight:bold" align="center" align="center" | 19 |style="font-weight:bold" align="center" align="center" valign="bottom" | 117.8 |} <li>Add 19 uL probe mix to each sample</li> <li>Incubate at 55C for 10 minutes</li> <li>Incubate at 25C for 10 minutes</li> <li>Lower the temperature to 20C; wait 30 seconds then take off incubator</li> <li>Add 1 uL T4 Ligase to each reaction</li> <li>Incubate at 16C overnight</li> Continued [[Daniel:Notebook/ComboLock/2017-6-15|tomorrow]] [[Category:ComboLock]] [[Category:20170614]]
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