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=emPCR 9-Concentration Comparison= The beads experiment above seemed to work, but I think the beads were a little under-loaded. I'm going to jack up the concentrations to make a new one. Hopefully this will give a bigger gradient of bead percentage loaded. ==Protocol== <ol> <li>Oil phase</li> <ol type="A"> <li>Mix 5 uL Triton X, 40 uL Tween-80, and 450 uL Span 80 into 2 mL Mineral oil and 8 mL Tegosoft DEC</li> <li>Vortex vigorously to mix</li> </ol> <li>Template-Bead Binding; Make '''3''' samples and follow this protocol for each</li> <ol type="A"> <li>Suspend 2.5 uL (25 ug, ~25M 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 10 uM (25 pmol total, 2X excess) dual biotin oligo and 15 uL nfH<sub>2</sub>O per sample to bead solution; incubate at RT for 15 min</li> </ol> <li>Aqueous Phase Preparation</li> <ol type="A"> <li>Wash beads with 100 uL wash buffer; vortex to suspend; apply magnet and remove supernatant</li> <li>Repeat wash step</li> <li>Resuspend beads in 10 uL TE buffer</li> <li>Mix ingredients using the following table</li> {| class="wikitable" <hiddentext>generated with [[:de:Wikipedia:Helferlein/VBA-Macro for EXCEL tableconversion]] V1.8</hiddentext> |- style="background-color:#CC66FF;font-size:12pt;font-weight:bold" align="center" | width="263" height="45" | Component | width="65" | uL Added | width="65" | 3.2X Master Mix uL Added | width="87" | Final Concentration |- style="font-size:12pt" | height="15" valign="bottom" | DreamTaq 10X PCR Buffer (20 mM MgCl2) | align="center" align="center" valign="bottom" | 10 | align="center" align="center" valign="bottom" | 32 | align="center" valign="bottom" | 1X |- style="background-color:#BFBFBF;font-size:12pt" | height="15" valign="bottom" | 10 mM dNTPs | align="center" align="center" valign="bottom" | 10 | align="center" align="center" valign="bottom" | 32 | align="center" valign="bottom" | 1 mM |- style="font-size:12pt" | height="15" valign="bottom" | 25 mM MgCl2 | align="center" align="center" valign="bottom" | 6 | align="center" align="center" valign="bottom" | 19.2 | align="center" valign="bottom" | 3.5 mM |- style="background-color:#BFBFBF;font-size:12pt" | height="15" valign="bottom" | 10 uM Primer A (Free primer-Primer 2) | align="center" align="center" valign="bottom" | 10 | align="center" align="center" valign="bottom" | 32 | align="center" valign="bottom" | 1 uM |- style="font-size:12pt" | height="15" valign="bottom" | 10 uM dual biotin primer | align="center" align="center" valign="bottom" | 0.5 | align="center" align="center" valign="bottom" | 1.6 | align="center" valign="bottom" | 0.1 uM |- style="background-color:#BFBFBF;font-size:12pt" | height="15" valign="bottom" | Hydrogel beads, 2M/uL | align="center" align="center" valign="bottom" | 10 | align="center" align="center" valign="bottom" | 32 | align="center" valign="bottom" | 25 M |- style="font-size:12pt" | height="15" valign="bottom" | DreamTaq Polymerase (5 U/uL) * | align="center" align="center" valign="bottom" | 3 | align="center" align="center" valign="bottom" | 0 | align="center" valign="bottom" | 15 Units |- style="background-color:#BFBFBF;font-size:12pt" | height="30" | Barcode Oligo v1 or v3 (template, 100 pM, 1 nM 10 nM) | align="center" align="center" | 10 | align="center" align="center" | 0 | align="center" | 10 pM, 100 pM, 1 nM |- style="font-size:12pt" | height="15" valign="bottom" | 100 mM Ammonium Sulfate | align="center" align="center" valign="bottom" | 5 | align="center" align="center" valign="bottom" | 16 | align="center" valign="bottom" | 5 mM |- style="background-color:#BFBFBF;font-size:12pt" | height="15" valign="bottom" | 1 mM Spermidine | align="center" align="center" valign="bottom" | 10 | align="center" align="center" valign="bottom" | 32 | align="center" valign="bottom" | 100 uM |- style="font-size:12pt" | height="15" valign="bottom" | Water | align="center" align="center" valign="bottom" | 25.5 | align="center" align="center" valign="bottom" | 81.6 | align="center" valign="bottom" | NA |- style="background-color:#BFBFBF;font-size:12pt" | height="15" valign="bottom" | Total Volume |style="font-weight:bold" align="center" align="center" valign="bottom" | 100 |style="font-weight:bold" align="center" align="center" valign="bottom" | 278.4 | align="center" align="center" valign="bottom" | |} <li>Add 2 uL DreamTaq Polymerase to each sample</li> </ol> <li>Oil-Aqueous Mixing</li> <ol type="A"> <li>Mix 10 uL aqueous into 100 uL oil mixture</li> <li>Vortex for 30 seconds</li> <li>Repeat mixing to 100 uL (5 times)</li> <li>Mix with vortexer for 3 minutes</li> <li>Dilute with pure mineral oil for imaging (1:100)</li> <li>Distribute 150 uL total volume into 3 tubes, 50 uL each; use same pipette tip</li> </ol> ****** <li>Emulsion PCR</li> <ol type="A"> <li>Perform PCR with the following settings</li> <ol type="a"> <li>95C 2 min</li> <li>95C 20 sec</li> <li>45C 30 sec</li> <li>70C 30 sec</li> <li>Goto b 95 times</li> <li>4C forever</li> </ol></ol> Continued [[Daniel:Notebook/PosSequencing/2017-7-13|tomorrow]] [[Category:PosSeq]] [[Category:20170706]] [[Category:20170712]]
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