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=emPCR 7-Strand Separation= [[Daniel:Notebook/PosSequencing|Back to Calendar]] Since [[Daniel:Notebook/PosSequencing/2017-6-27|yesterday]]'s results showed DNA in the two fractions (10 nM and 10 pM) after emulsion breaking but before adding the alkaline buffer for strand separation. Therefore, I'll try again but I'll split the fractions post-emulsion breaking and use several different pH values to test effectiveness. ==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 4 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="290" height="30" | Component | width="95" | uL Added | width="95" | uL Added | width="120" | Final Concentration |- style="font-size:12pt" | height="15" valign="bottom" | DreamTaq 10X PCR Buffer (20 mM MgCl2) | align="right" align="center" valign="bottom" | 10 | align="right" align="center" valign="bottom" | 10 | align="center" valign="bottom" | 1X |- style="background-color:#BFBFBF;font-size:12pt" | height="15" valign="bottom" | 10 mM dNTPs | align="right" align="center" valign="bottom" | 10 | align="right" align="center" valign="bottom" | 10 | align="center" valign="bottom" | 1 mM |- style="font-size:12pt" | height="15" valign="bottom" | 25 mM MgCl2 | align="right" align="center" valign="bottom" | 6 | align="right" align="center" valign="bottom" | 6 | 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="right" align="center" valign="bottom" | 10 | align="right" align="center" valign="bottom" | 10 | align="center" valign="bottom" | 1 uM |- style="font-size:12pt" | height="15" valign="bottom" | 10 uM dual biotin primer | align="right" align="center" valign="bottom" | 0.5 | align="right" align="center" valign="bottom" | 0.5 | align="center" valign="bottom" | 0.1 uM |- style="background-color:#BFBFBF;font-size:12pt" | height="15" valign="bottom" | Hydrogel beads, 2M/uL | align="right" align="center" valign="bottom" | 10 | align="right" align="center" valign="bottom" | 10 | align="center" valign="bottom" | 25 M |- style="font-size:12pt" | height="15" valign="bottom" | DreamTaq Polymerase (5 U/uL) * | align="right" align="center" valign="bottom" | 2 | align="right" align="center" valign="bottom" | 2 | align="center" valign="bottom" | 10 Units |- style="background-color:#BFBFBF;font-size:12pt" | height="15" | Barcode Oligo v1 (template, 100 pM, 1 pM*) | align="right" align="center" | 5 | align="center" | 5* | align="center" valign="bottom" | 10 pM, 0.1 pM* |- style="font-size:12pt" | height="15" valign="bottom" | 100 mM Ammonium Sulfate | align="right" align="center" valign="bottom" | 5 | align="right" align="center" valign="bottom" | 5 | align="center" valign="bottom" | 5 mM |- style="background-color:#BFBFBF;font-size:12pt" | height="15" valign="bottom" | 1 mM Spermidine*** | align="right" align="center" valign="bottom" | 10 | align="right" align="center" valign="bottom" | 10 | align="center" valign="bottom" | 100 uM |- style="font-size:12pt" | height="15" valign="bottom" | TIPP | align="right" align="center" valign="bottom" | 0 | align="right" align="center" valign="bottom" | 0 | align="right" align="center" valign="bottom" | 0 |- style="background-color:#BFBFBF;font-size:12pt" | height="15" valign="bottom" | Water | align="right" align="center" valign="bottom" | 36.5 | align="right" align="center" valign="bottom" | 36.5 | align="center" valign="bottom" | NA |- style="font-size:12pt" | height="15" valign="bottom" | Total Volume |style="font-weight:bold" align="right" align="center" valign="bottom" | 100 |style="font-weight:bold" align="right" align="center" valign="bottom" | 100 | align="right" align="center" valign="bottom" | |} *Note: The 1 pM sample is the concentration suggested to use. This will help me know about how many cycles to use for production in the future </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>58C 30 sec</li> <li>70C 30 sec</li> <li>Goto b 95 times</li> <li>4C forever</li> </ol></ol> <li>Prepare Water saturated compounds</li> <ol type="A"> <li>Mix 5 mL diethyl ether and 5 mL ddH2O in a falcon tube; allow to settle, extract top (ether) phase</li> <li>Mix 5 mL ethyl acetate and 5 mL ddH2O in a falcon tube; allow to settle, extract top (acetate) phase</li> </ol> <li>Emulsion Breaking</li> <ol type="A"> <li>With a 200 uL pipette add 100 uL diethyl ether to each sample; save the tip</li> <li>Adjust tip to 200 and using same tip pool samples in a new 1.5 mL tube</li> <li>Vortex the pooled sample in the 1.5 mL tube for 30 seconds</li> <li>Spin down the sample 13,000 rpm for 5 minutes</li> <li>Take off oil-DE mix (top layer) but do not disrupt the oil/aqueous interface</li> <li>Add 1000 uL ethyl acetate and vortex for 30 seconds</li> <li>Spin down sample at 13,000rpm for 3 minutes</li> <li>Remove ethyl acetate without disturbing the pellet</li> <li>Add 1000 uL diethyl ether and vortex for 30 seconds</li> <li>Spin down sample at 13,000rpm for 3 minutes</li> <li>Remove DE without disturbing the aqueous phase</li> <li>Allow DE to dry in the fume hood; <span style="background:#FFFF00">resuspend beads in the solution and take 15 uL aside for testing</span></li> </ol> </ol> Continued [[Daniel:Notebook/PosSequencing/2017-6-29|tomorrow]] [[Category:PosSeq]] [[Category:20170628]]
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