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=emPCR 7-Strand Separation (Started [[Daniel:Notebook/PosSequencing/2017-6-28|Wednesday June 28]])= [[Daniel:Notebook/PosSequencing|Back to Calendar]] ==Protocol== <ol> <li>Template-bead binding; do this twice, once with no template</li> <ol type="A"> <li>Suspend 1 uL (10 ug, ~10M beads) beads in 100 uL Dynabuffer</li> <li>Apply magnet for 30 sec and remove supernatant</li> <li>Suspend in 20 uL Dynabuffer (conc. 2.5 ug/uL)</li> <li>Add 5 uL 500 nM (25 pmol total, 0.5X excess) dsDNA and 15 uL nfH<sub>2</sub>O per sample to bead solution; incubate at RT for 15 min; for NT sample add 20 dynabuffer/20 nfH<sub>2</sub>O</li> <li>Wash twice with 100 uL dynabuffer</li> <li>Resuspend beads in 50 uL TE buffer</li> </ol> <li>DRAQ5 staining</li> <ol>Take 5 uL aliquots and add 43 uL nfH<sub>2</sub>O and 2 uL DRAQ5 (1:10000)</li> </ol> <li>Slide preparation</li> <ol type="A"> <li>Add 15 uL of aliquot to a slide</li> <li>Add a #1.5 coverslip and seal with nail polish; wait approximately 20 minutes before imaging</li> </ol></ol> ==Results== <gallery perrow=3 heights=300px widths=300px mode=packed-hover caption="DRAQ5 DNA staining for Blank Beads"> File:20170705-PosSequencing-emPCR7.lif_BlankBeads-Field1-SNAP_ch00.jpg|Field 1 File:20170705-PosSequencing-emPCR7.lif_BlankBeads-Field2-SNAP_ch00.jpg|Field 2 File:20170705-PosSequencing-emPCR7.lif_BlankBeads-Field3-SNAP_ch00.jpg|Field 3 </gallery> <gallery perrow=3 heights=300px widths=300px mode=packed-hover caption="DRAQ5 DNA staining for dsDNA"> File:20170705-PosSequencing-emPCR7.lif_dsDNA-Field1-SNAP_ch00.jpg|Field 1 File:20170705-PosSequencing-emPCR7.lif_dsDNA-Field2-SNAP_ch00.jpg|Field 2 File:20170705-PosSequencing-emPCR7.lif_dsDNA-Field3-SNAP_ch00.jpg|Field 3 </gallery> <gallery perrow=3 heights=300px widths=300px mode=packed-hover caption="DRAQ5 DNA staining for 100pM starting material"> File:20170705-PosSequencing-emPCR7.lif_100pM-Field1-SNAP_ch00.jpg|Field 1 File:20170705-PosSequencing-emPCR7.lif_100pM-Field2-SNAP_ch00.jpg|Field 2 File:20170705-PosSequencing-emPCR7.lif_100pM-Field3-SNAP_ch00.jpg|Field 3 </gallery> <gallery perrow=3 heights=300px widths=30px mode=packed-hover caption="DRAQ5 DNA staining for 1 pM starting template"> File:20170705-PosSequencing-emPCR7.lif_1pM-Field1-SNAP_ch00.jpg|Field 1 File:20170705-PosSequencing-emPCR7.lif_1pM-Field2-SNAP_ch00.jpg|Field 2 File:20170705-PosSequencing-emPCR7.lif_1pM-Field3-SNAP_ch00.jpg|Field 3 </gallery> ==Discussion== It's not exactly easy to spot unless you have a fancy computer, so here's the gist. The '''blank beads samples''' show that without DNA the DRAQ5 doesn't stain the beads, and there isn't enough autofluorescence at that wavelength to look like much. When you do max projection (which all of these are) and adjust the thresholds to similar values as with the samples, the resulting signal is not next to the dots. The '''dsDNA positive control''' on the other hand, has the signal overlap with the beads, although not on all samples. This makes sense, however, as those beads are generally under packed (50% of theoretical maximum was added), meaning a simple Poissonian predicts that a lot of them will have little signal. Some, however, will and do have the signal. The '''100 pM samples''' have good signal, although I will note that on the samples the beads tend to stick together more often. The '''1 pM''' samples on the other hand, show no real signal. So the 1 pM sample did not have enough amplification. [[Category:20170628]] [[Category:PosSeq]]
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