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=Beadless emPCR (Started [[Daniel:Notebook/PosSequencing/2017-6-22|Thursday 6/22/17]])= [[Daniel:Notebook/PosSequencing|Back to calendar]] Since the results on [[Daniel:Notebook/PosSequencing/2017-6-23|Friday]] showed viable product (at least in the 100 nM sample), I'm going to finish up that experiment with strand denaturation and single primer qPCR. ==Protocol== <ol start="9"> <li>Strand Separation (DNA Denaturation)</li> <ol type="A"> <li>Add 500 uL KOH buffer (pH 12.0)</li> <li>Spin down sample at 13,000rpm for 1 minute</li> <li>Remove all but 40uL buffer</li> <li>Repeat KOH steps A-C once more</li> <li>Add 500 uL 4X SSC buffer (pH 12.0)</li> <li>Spin down sample at 13,000rpm for 1 minute</li> <li>Remove all but 10uL buffer</li> <li>Resuspend in 80 uL 2X SSC buffer</li> </ol> <li>Single primer qPCR</li> <ol type="A"> <li>Make the following master mixes</li> {| 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="210" height="30" | Reagent | width="95" | uL in Single Rxn | width="95" | Master Mix P2 (4.2X) | width="95" | Master Mix BioU (4.2X) | width="95" | Master Mix Dual (6.2X) |- style="font-size:12pt" | height="15" valign="bottom" | Primer 2 (10 uM) | align="center" align="center" valign="bottom" | 1 | align="center" align="center" valign="bottom" | 4.2 | align="center" align="center" valign="bottom" | 0 | align="center" align="center" valign="bottom" | 6.2 |- style="background-color:#BFBFBF;font-size:12pt" | height="15" valign="bottom" | BioU Primer (10 uM) | align="center" align="center" valign="bottom" | 1 | align="center" align="center" valign="bottom" | 0 | align="center" align="center" valign="bottom" | 4.2 | align="center" align="center" valign="bottom" | 6.2 |- style="font-size:12pt" | height="15" valign="bottom" | 2X Kapa SYBR Master Mix | align="center" align="center" valign="bottom" | 20 | align="center" align="center" valign="bottom" | 84 | align="center" align="center" valign="bottom" | 84 | align="center" align="center" valign="bottom" | 124 |- style="background-color:#BFBFBF;font-size:12pt" | height="15" valign="bottom" | nfH2O | align="center" align="center" valign="bottom" | 16 | align="center" align="center" valign="bottom" | 67.2 | align="center" align="center" valign="bottom" | 67.2 | align="center" align="center" valign="bottom" | 93 |- style="font-size:12pt" | height="15" valign="bottom" | Total* (+ 5 uL sample=40) |style="font-weight:bold" align="center" align="center" valign="bottom" | 35 |style="font-weight:bold" align="center" align="center" valign="bottom" | 155.4 |style="font-weight:bold" align="center" align="center" valign="bottom" | 155.4 |style="font-weight:bold" align="center" align="center" valign="bottom" | 229.4 |} <li>Aliquot 35 uL master mix to appropriate lanes</li> <li>Add 2 uL sample according to plate layout below</li> [[Image:PlateLayout-Beadless-emPCR-singleprimer-20170626.png|522x348px]] <li>Run the following thermocycler program</li> <ol type="a"> <li>95C 3 min</li> <li>95C 3 sec</li> <li>45C 30 sec</li> <li>72C 20 sec</li> <li>plate read</li> <li>goto b x90</li> <li>72C 2 min</li> <li>16C hold</li> </ol></ol> </ol> ==Results== <gallery perrow=2 heights=250px widths=350px mode=packed-hover> File:PlateLayout-Beadless-emPCR-singleprimer-20170626.png|Plate layout File:Beadless-emPCR-singleprimer-20170626-plateCTs-normed.png|Plate CTs File:Beadless-emPCR-singleprimer-20170626-rawcurves.png|Raw curves </gallery> =emPCR 6= Based on the results from [[Daniel:Notebook/PosSequencing/2017-6-23|Friday]] and continued above, I think this means I'm clear to try again on emPCR using the lower annealing temp. I'll give 15 more cycles since the pM sample didn't amplify. ==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, 100 nM) | align="right" align="center" | 5 | align="center" | 5* | align="center" valign="bottom" | 10 pM, 10 nM* |- 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" | |} </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> </ol> Continued [[Daniel:Notebook/PosSequencing/2017-6-27|tomorrow]] [[Category:PosSeq]] [[Category:20170622]] [[Category:20170626]]
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