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Daniel:Notebook/ComboLock/2017-6-16
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=Antibody Detection Test-Positive Control (Started [[Daniel:Notebook/ComboLock/2017-6-14|Wednesday]])= [[Daniel:Notebook/ComboLock|Back to Calendar]] ==Protocol-Part 5 (From [[Daniel:Notebook/ComboLock/2017-6-15|Yesterday]])== <ol start="9"> <li>Bglii digestion</li> <ol type="A"> <li>Make the following reactions; '''Do not add Bglii yet'''</li> {| class="wikitable" <hiddentext>generated with [[:de:Wikipedia:Helferlein/VBA-Macro for EXCEL tableconversion]] V1.8</hiddentext> |- style="font-size:12pt;font-weight:bold" align="center" |style="background-color:#8DB4E2" width="145" height="30" | Reagent |style="background-color:#8DB4E2" width="95" | Single Rxn Vol |style="background-color:#95B3D7" width="95" | Master Mix (6.2X) |- style="font-size:12pt" | height="15" valign="bottom" | RCA Rxn | align="center" align="center" valign="bottom" | 7 | align="center" align="center" valign="bottom" | 0 |- style="background-color:#BFBFBF;font-size:12pt" | height="15" valign="bottom" | 10X Buffer 3.1 | align="center" align="center" valign="bottom" | 2 | align="center" align="center" valign="bottom" | 12.4 |- style="font-size:12pt" | height="15" valign="bottom" | Bglii | align="center" align="center" valign="bottom" | 1 | align="center" align="center" valign="bottom" | 6.2 |- style="background-color:#BFBFBF;font-size:12pt" | height="15" valign="bottom" | nfH2O | align="center" align="center" valign="bottom" | 10 | align="center" align="center" valign="bottom" | 62 |- style="font-size:12pt" | height="15" valign="bottom" | Total |style="font-weight:bold" align="center" align="center" valign="bottom" | 20 |style="font-weight:bold" align="center" align="center" valign="bottom" | 80.6 |} <li>Aliquot 12 uL master mix into new tube</li> <li>Add 7 uL appropriate sample</li> <li>Mix with vortexer, spin down, and add 1 uL Bglii</li> <li>Incubate 2 hr 37C</li> <li>Heat kill with 65C for 20 min</li> <li>Hold at 12C</ol> <li>qPCR</li> <ol type="A"> <li>Make the following 7.2X master mix (1X)</li> <ol type="a"> <li>7.2 uL 10 uM AmpF6.4Sol</li> <li>151.2 uL (21) nfH<sub>2</sub>O</li> <li>180 uL 2X Kapa SYBR Fast Master Mix</li> </ol> <li>Add 47 uL master mix to each lane</li> <li>Add 1 uL AmpR and 2 uL sample according to plate layout</li> [[Image:PlateLayout-20170616-AntibodyTest.png|350x250px]] <li>Use the following protocol</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 x30</li> <li>72C 2 min</li> <li>16C hold</li> </ol></ol> <li>TBE Gel</li> <ol type="A"> <li>Mix together 120 uL TBE and 30 uL 6X dye</li> <li>Aliquot 10 uL/lane onto parafilm</li> <li>Add 2 uL correct sample or (1.5 uL for ladder) to aliquots</li> <li>Add 10 uL aliquots to correct lanes</li> <li>Run gel at 235V for 24 minutes</li> <li>Remove gel from case and incubate in TBE buffer with 2 uL SYBR Gold for 3 minutes</li> <li>Image in gel doc</li> </ol> </ol> ==Results== <gallery perrow=2 heights=250px widths=300px mode=packed-hover> File:PlateLayout-20170616-AntibodyTest.png|Plate layout File:Antibodytest-controlsystem-20170615-plateCTs-normed.png|Plate CTs File:Antibodytest-controlsystem-20170615-rawcurves.png|Raw curves File:2017-06-19-AntibodyTest-ControlSystem.png|Gel image </gallery> [[Category:ComboLock]] [[Category:20170614]]
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