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=Antibody Hybridization Test= [[Daniel:Notebook/ComboLock|Back to Calendar]] Time to test out the antibody binding protocol like I did with [[Daniel:Notebook/ComboLock/2016-5-24|C-probes]] ==Protocol== <ol> <li>Mix PLA Buffer</li> {| class="wikitable" <hiddentext>generated with [[:de:Wikipedia:Helferlein/VBA-Macro for EXCEL tableconversion]] V1.8</hiddentext> |- style="background-color:#D8E4BC;font-size:12pt" align="center" | width="118" height="30" | Reagent | width="101" | Stock Conc. | width="65" | Final Conc. | width="65" | Dilution | width="88" | Volume for 1 mL solution (uL) |- style="font-size:12pt" | height="15" valign="bottom" | BSA* | align="center" valign="bottom" | 50 mg/mL (5%) | align="center" align="center" valign="bottom" | 0.10% | align="center" align="center" valign="bottom" | 50 | align="center" align="center" valign="bottom" | 20 |- 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" | 0.05% | align="center" align="center" valign="bottom" | 2000 | align="center" align="center" valign="bottom" | 0.5 |- style="font-size:12pt" | height="15" valign="bottom" | Salmon Sperm DNA | align="center" valign="bottom" | 10 mg/mL | align="center" valign="bottom" | 0.1 mg/mL | align="center" align="center" valign="bottom" | 100 | align="center" align="center" valign="bottom" | 10 |- style="background-color:#D9D9D9;font-size:12pt" | height="15" valign="bottom" | EDTA | align="center" valign="bottom" | 500 mM | align="center" valign="bottom" | 5 mM | align="center" align="center" valign="bottom" | 100 | align="center" align="center" valign="bottom" | 10 |- 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" align="center" valign="bottom" | 100 |- style="background-color:#D9D9D9;font-size:12pt" | height="15" valign="bottom" | nfH2O | align="center" valign="bottom" | -------- | align="center" valign="bottom" | -------- | align="center" valign="bottom" | -------- | align="center" align="center" valign="bottom" | 859.5 |} <li>Hybridization</li> <ol type="A"> <li>Split [[Daniel:Notebook/ComboLock/2016-5-28|methanol-frozen]] cells into 4 (1.25 mL methanol each)</li> <li>Pellet cells via centrifugation at 600g for 3 min</li> <li>Add PLA mix and probes to create hybridization solution</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="30" | |style="background-color:#CCC0DA;font-weight:bold" width="71" | uL PA Mix |style="background-color:#CCC0DA;font-weight:bold" width="75" | uL PB Mix |style="background-color:#CCC0DA;font-weight:bold" width="79" | uL PLA buffer |- style="font-size:12pt" align="center" | height="15" | Sample 1 | align="center" | 0.5 | align="center" | 0 | align="center" | 2.5 |- style="background-color:#D9D9D9;font-size:12pt" align="center" | height="15" | Sample 2 | align="center" | 0 | align="center" | 0.5 | align="center" | 2.5 |- style="font-size:12pt" align="center" | height="15" | Sample 3 | align="center" | 0.5 | align="center" | 0.5 | align="center" | 2 |- style="background-color:#D9D9D9;font-size:12pt" align="center" | height="15" | Sample 4 | align="center" | 0 | align="center" | 0 | align="center" | 3 |} <li>Mix 1 uL cell lysate with 3 uL Probe mix</li> <li>Incubate 1.5 hours at RT</li> <li>Add 200 uL 0.05% Tween 20 in PBS</li> <li>Wash twice with 0.05% Tween 20 in PBS</li> <ol type="a"><li>Pellet cells at 600xg for 3 min</li></ol> <li>Resuspend samples in 50 uL 1X PBS</li> </ol> <li>qPCR</li> <ol type="A"> <li>Set up qPCR reaction according to table</li> {| class="wikitable" <hiddentext>generated with [[:de:Wikipedia:Helferlein/VBA-Macro for EXCEL tableconversion]] V1.8</hiddentext> |- style="background-color:#FCD5B4;font-size:12pt;font-weight:bold" align="center" | width="120" height="45" | Sample | width="65" | Lane | width="65" | Cell Sample | width="65" | Cells | width="65" | 2X Kapa SYBR qPCR MM | width="65" | 100 uM Primer2 | width="65" | 100 uM Primer4-RC | width="65" | 100 uM Primer6 | width="65" | 100 uM Primer12-RC | width="65" | H2O | width="65" | Total Volume (uL) |- style="font-size:12pt" | height="15" valign="bottom" | Sample 1 (PA) | align="center" valign="bottom" | A1 | align="center" valign="bottom" | S1 | align="center" align="center" valign="bottom" | 1 | align="center" align="center" valign="bottom" | 25 | align="center" align="center" valign="bottom" | 0.5 | align="center" align="center" valign="bottom" | 0.5 | align="center" align="center" valign="bottom" | 0 | align="center" align="center" valign="bottom" | 0 | align="center" align="center" valign="bottom" | 23 | align="center" align="center" valign="bottom" | 50 |- style="background-color:#D9D9D9;font-size:12pt" | height="15" valign="bottom" | Sample 2 (PB) | align="center" valign="bottom" | A2 | align="center" valign="bottom" | S2 | align="center" align="center" valign="bottom" | 1 | align="center" align="center" valign="bottom" | 25 | align="center" align="center" valign="bottom" | 0 | align="center" align="center" valign="bottom" | 0 | align="center" align="center" valign="bottom" | 0.5 | align="center" align="center" valign="bottom" | 0.5 | align="center" align="center" valign="bottom" | 23 | align="center" align="center" valign="bottom" | 50 |- style="font-size:12pt" | height="30" valign="bottom" | Sample 3 (PA+PB) | align="center" valign="bottom" | A3 | align="center" valign="bottom" | S3 | align="center" align="center" valign="bottom" | 1 | align="center" align="center" valign="bottom" | 25 | align="center" align="center" valign="bottom" | 0.5 | align="center" align="center" valign="bottom" | 0.5 | align="center" align="center" valign="bottom" | 0.5 | align="center" align="center" valign="bottom" | 0.5 | align="center" align="center" valign="bottom" | 22 | align="center" align="center" valign="bottom" | 50 |- style="background-color:#D9D9D9;font-size:12pt" | height="30" valign="bottom" | Sample 4 (No Probe) | align="center" valign="bottom" | A4 | align="center" valign="bottom" | S4 | align="center" align="center" valign="bottom" | 1 | align="center" align="center" valign="bottom" | 25 | align="center" align="center" valign="bottom" | 0.5 | align="center" align="center" valign="bottom" | 0.5 | align="center" align="center" valign="bottom" | 0.5 | align="center" align="center" valign="bottom" | 0.5 | align="center" align="center" valign="bottom" | 22 | align="center" align="center" valign="bottom" | 50 |- style="font-size:12pt" | height="15" valign="bottom" | No Template | align="center" valign="bottom" | A5 | align="center" valign="bottom" | NA | align="center" align="center" valign="bottom" | 0 | align="center" align="center" valign="bottom" | 25 | align="center" align="center" valign="bottom" | 0.5 | align="center" align="center" valign="bottom" | 0.5 | align="center" align="center" valign="bottom" | 0.5 | align="center" align="center" valign="bottom" | 0.5 | align="center" align="center" valign="bottom" | 23 | align="center" align="center" valign="bottom" | 50 |- style="background-color:#D9D9D9;font-size:12pt" | height="30" valign="bottom" | Mismatch Primers | align="center" valign="bottom" | A6 | align="center" valign="bottom" | S3 | align="center" align="center" valign="bottom" | 1 | align="center" align="center" valign="bottom" | 25 | align="center" align="center" valign="bottom" | 0.5 | align="center" align="center" valign="bottom" | 0 | align="center" align="center" valign="bottom" | 0.5 | align="center" align="center" valign="bottom" | 0 | align="center" align="center" valign="bottom" | 23 | align="center" align="center" valign="bottom" | 50 |- style="font-size:12pt" | height="15" valign="bottom" | No Primers | align="center" valign="bottom" | A7 | align="center" valign="bottom" | S3 | align="center" align="center" valign="bottom" | 1 | align="center" align="center" valign="bottom" | 25 | align="center" align="center" valign="bottom" | 0 | align="center" align="center" valign="bottom" | 0 | align="center" align="center" valign="bottom" | 0 | align="center" align="center" valign="bottom" | 0 | align="center" align="center" valign="bottom" | 24 | align="center" align="center" valign="bottom" | 50 |- style="background-color:#D9D9D9;font-size:12pt" | height="30" valign="bottom" | Primers + Antibodies | align="center" valign="bottom" | A8 | align="center" valign="bottom" | NA | align="center" align="center" valign="bottom" | 1 | align="center" align="center" valign="bottom" | 25 | align="center" align="center" valign="bottom" | 0.5 | align="center" align="center" valign="bottom" | 0.5 | align="center" align="center" valign="bottom" | 0.5 | align="center" align="center" valign="bottom" | 0.5 | align="center" align="center" valign="bottom" | 22 | align="center" align="center" valign="bottom" | 50 |} <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 x20</li> <li>72C 2 min</li> <li>16C hold</li> </ol> </ol> ==qPCR Results== <gallery perrow=2 heights=300px widths=300px> File:VIM-AbTest1_Results_20160602.png|Results w/o labeling File:20160602-Antibody_Test_qPCR_Curve.png|Results with labeling </gallery>
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