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=emPCR8 (Started [[Daniel:Notebook/PosSequencing/2017-7-6|Thursday July 6]])= [[Daniel:Notebook/PosSequencing|Back to Calendar]] ==PolyT Primer qPCR== The purpose of this slight deviation is to see if using the PolyT primer (which adds another 22 bases) to the final sequence produces an NTC band that is distinct from the product band. <ol> <li>qPCR</li> <ol type="A"> <li>Dilute 2 uL each 10 uM 2-Biotin-Uracil and Primer2 into 16 uL nfH<sub>2</sub>O (1:10)</li> <li>Prepare the following 4.2X (1X) master mix</li> <ol type="a"><li>16.8 uL 1 uM primers (4, 2 each primer; conc 0.05 uM)</li><li>58.8 (16) uL nfH<sub>2</sub>O</li><li>84 uL 2X Kapa SYBR master mix</li></ol> <li>Aliquot 38 uL master mix into each lane</li> <li>Add 2 uL appropriate sample according to plate layout</li> [[Image:PlateLayout20170711-emPCR8-PolyT.png|350x250px]] <li>Run the single primer protocol (main difference is 45C melting temp</li> for ~40 cycles</li> </ol> <li>TBE Gel</li> <ol type="A"> <li>Mix together 64 uL 1X TBE and 16 uL 6X loading dye</li> <li>Aliquot 10 uL per sample onto parafilm</li> <li>Add 1.5 uL 25bp ladder or 2 uL sample to appropriate lanes</li> <li>Mix and add 10 uL to appropriate gel wells</li> <li>Run gel for 25 minutes at 235V</li> <li>Open gel and stain with 2 uL SYBR gold for 3 minutes</li> <li>Rinse once and image in gel doc</li> </ol> </ol> ==qPCR Results== <gallery perrow=2 heights=250px widths=350px mode=packed-hover> File:PlateLayout20170711-emPCR8-PolyT.png|Plate layout File:EmPCR8-20170711-PolyT-plateCTs-normed.png|Plate CTs File:EmPCR8-20170711-PolyT-rawcurves.png|Raw curves File:|Gel image </gallery> ==Repeated Protocol== Since I still have ~20 uL left of the bead mix, I'll use a lot of that to try the experiment again. <ol start="7"> <li>Phosphorylation</li> <ol type="A"> <li>Pull down beads, remove supernatant, and wash with 100 uL DynaT-buffer (Dynabuffer + 0.05% Tween20)</li> <li>Pull down beads and remove supernatant</li> <li>Resuspend the beads in the following mixture</li> {| class="wikitable" <hiddentext>generated with [[:de:Wikipedia:Helferlein/VBA-Macro for EXCEL tableconversion]] V1.8</hiddentext> |- style="background-color:#C4D79B;font-size:12pt;font-weight:bold" align="center" | width="200" height="42" | Reagent | width="95" | uL 1X Reaction | width="95" | uL 3.2X Master Mix |- style="font-size:12pt" | height="15" valign="bottom" | T4 PNK Buffer (10X) | align="right" align="center" valign="bottom" | 5 | align="right" align="center" valign="bottom" | 16 |- style="background-color:#BFBFBF;font-size:12pt" | height="15" valign="bottom" | ATP (10 mM) | align="right" align="center" valign="bottom" | 5 | align="right" align="center" valign="bottom" | 16 |- style="font-size:12pt" | height="15" valign="bottom" | T4 PNK (10 U/uL) | align="right" align="center" valign="bottom" | 1 | align="right" align="center" valign="bottom" | 3.2 |- style="background-color:#BFBFBF;font-size:12pt" | height="15" valign="bottom" | nfH2O | align="right" align="center" valign="bottom" | 39 | align="right" align="center" valign="bottom" | 124.8 |- style="font-size:12pt" | height="15" valign="bottom" | Total |style="font-weight:bold" align="right" align="center" valign="bottom" | 50 |style="font-weight:bold" align="right" align="center" valign="bottom" | 160 |} <li>Resuspend each sample in 49 uL master mix</li> <li>Add 1 uL T4 PNK to each</li> <li>Incubate for 1 minutes at 37C</li> <li>Heat inactivate by incubating for 20 minutes at 65C</li> </ol> <li>Lambda Exonuclease Digestion</li> <ol type="A"> <li>Pull down with magnet and remove supernatant</li> <li>Make the following master mix</li> <ol type="a"><li>16 uL 10X Lambda exonuclease reaction buffer</li><li>140.8 uL nfH<sub>2</sub>O</li></ol> <li>Resuspend beads in 49 uL master mix</li> <li>Add 1 uL lambda exonuclease (10 U/uL)</li> <li>Incubate for 30 minutes at 37C</li> <li>Heat kill enzyme by incubating for 10 minutes at 80C</li> <li>Magnet pulldown, and resuspend in 50 uL 2XSSC</li> </ol> </ol> Continued [[Daniel:Notebook/PosSequencing/2017-7-12|tomorrow]] [[Category:PosSeq]] [[Category:20170706]]
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