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=Multiplex Experiment (Started [[Daniel:Notebook/ComboLock/2017-7-13|Thursday July 13]])= [[Daniel:Notebook/ComboLock|Back to Calendar]] ==Protocol-Part 4 (Continued from [[Daniel:Notebook/ComboLock/2017-7-14|Friday July 14]])== <ol start="9"> <li>Bglii digestion</li> <ol type="A"> <li>Make the following reactions; '''Do not add Bglii yet'''</li> {| class="wikitable" |- style="font-size:12pt;font-weight:bold" align="center" |style="background-color:#8DB4E2" width="170" height="30" | Reagent |style="background-color:#8DB4E2" width="100" | Single Rxn Vol |style="background-color:#95B3D7" width="100" | Master Mix (4.2X) |- 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" | 8.4 |- style="font-size:12pt" | height="15" valign="bottom" | Bglii | align="center" align="center" valign="bottom" | 1 | align="center" align="center" valign="bottom" | 0 |- 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" | 42 |- 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" | 50.4 |} <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 5.2X master mix according to following (1X)</li> <ol type="a"> <li>109.2 uL nfH<sub>2</sub>O (21)</li> <li>5.2 uL 10 uM AmpF6.4Sol (1)</li> <li>130 uL 2X Kapa SYBR Fast Master Mix (25)</li></ol> <li>Add 47 uL master mix to each well</li> <li>Add 2 uL sample and 1 uL AmpR Index (10 uM) according to plate layout</li> [[Image:PlateLayout20170717-MultiplexExperiment.png|250x350px]] <li>Run the [[Daniel:Protocols/qPCR|Sstandard qPCR protocol]] for ~30 cycles</li> </ol> <li>TBE Gel</li> <ol type="A"> <li>Mix together 64 uL TBE and 16 uL 6X dye</li> <li>Aliquot 10 uL/lane onto parafilm</li> <li>Add 2 uL correct sample or 1.5 uL 25bp 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=350px mode=packed-hover> File:PlateLayout20170717-MultiplexExperiment.png|Plate layout File:Multiplex-20170717-plateCTs-normed.png|Plate CTs File:Multiplex-20170717-rawcurves.png|Raw curves File:2017-07-17-MultiplexComboLock.png|Gel image </gallery> [[Category:ComboLock]] [[Category:20170713]]
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