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=Bead Binding Test 2= [[Daniel:Notebook/PosSequencing|Back to Calendar]] The experiment is building from the confusing results from [[Daniel:Notebook/PosSequencing/2017-5-8|Monday]]. Basically I can't tell if the hybridization of the dye probes worked or not. The signal is just too low. This experiment will test oligo binding using DAPI, which should give clear results for dsDNA (the original molecule) vs ssDNA (after melting). ==Protocol== '''Sample Matrix''' {| 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="85" height="30" | Sample # | width="250" | Condition |- style="font-size:12pt" align="center" valign="bottom" | align="center" height="15" | 1 | Barcode Oligo 2 |- style="font-size:12pt" align="center" valign="bottom" | align="center" height="15" | 2 | Barcode Oligo 2-No Melting |} <ol> <li>Bead Binding</li> <ol type="A"> <li>Suspend 1 uL (10 ug, ~5M beads designed at 2.5 pmol) beads in 100 uL Dynabuffer</li> <li>Apply magnet for 30 sec and remove supernatant</li> <li>Suspend in 10 uL Dynabuffer (conc. 1 ug/uL)</li> <li>Add 5 uL barcode oligo and up to 5 uL nfH<sub>2</sub>O per sample to bead solution; incubate at RT for 15 min</li> <li>Wash beads with 100 uL wash buffer; vortex to suspend; apply magnet and remove supernatant</li> <li>Add 100 uL wash buffer and 1 uL DAPI; incubate in the dark for 5 minutes;</li> <li>Add 15 uL to a slide for analysis</li> <li>Remove the rest of the supernatant after bead pulldown</li> </ol> <li>Melting</li> <ol type="A"> <li>Resuspend in 100μL of TE-Triton 0.1% (TE-T)</li> <li>Melt off the complementary DNA strand by incubating the sample at 95°C for 3 minutes</li> <li>Magnet pull-down for 1 minute and remove supernatant</li> <li>Add 100 μL of TE-T and mix; Magnet pulldown and remove supernatant</li> <li>Add 100 μL of TE-T and mix</li> <li>Add 15 uL to a slide for analysis</li> <li>Remove the rest of the supernatant after bead pulldown</li> <li>Hold here and wait for results from slides</li> </ol> <li>Fluorescent Oligo Binding (All samples)</li> <ol type="A"> <li>Resuspend beads in 25 uL 4X SSC buffer</li> <li>Make the following 3.1X (1X) dye hybridization buffer</li> <ol type="a"> <li>6.2 (2 ea; 6 total) each uL 10 uM dye; dcprobe6-488, dcprobe6-Cy3, dcprobe6-Cy5</li> <li>46.5 (15) uL formamide</li> <li>12.4 (4) uL nfH<sub>2</sub>O</li></ol> <li>Incubate probes at 80C for 5 minutes</li> <li>Add 25 uL fluorescent probe in 60% formamide to each sample</li> <li>Incubate at room temp in the dark for 30 min</li> <li>Wash twice in 2X SSC</li> <li>Resuspend in 40 uL 2XSSC or 20 uL 2X SSC</li> </ol> </ol> [[Category:PosSeq]] [[Category:20170508]]
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