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=Emulsion PCR Test 2= [[Daniel:Notebook/PosSequencing|Back to Calendar]] From the results on ==Protocol== <ol> <li>Oil Production</li> <ol type="A"> <li>Start from [[Daniel:Notebook/PosSequencing/2017-4-10|Monday April 10]]'s oil prep (Span80-4.5%, Tween20-0.1%, TritonX100-0.05% in mineral oil)</li></ol> <li>Template-Bead Binding; Make 4 samples and follow this protocol for each</li> <ol type="A"> <li>Suspend 5 uL (50 ug, ~50M 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 2 uL 1 uM (10 pmol total, 2X excess) template oligo and 18 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>Repeat wash step</li> <li>Resuspend beads in 10 uL TE buffer</li> <li>Mix together the three suspensions of beads with different oligos</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="340" height="45" | Component | width="85" | Ion Torrent Concentration | width="110" | My uL Added- Sample (Single) | width="180" | My Final Concentration |- style="font-size:12pt" | height="15" valign="bottom" | DreamTaq 10X PCR Buffer (20 mM MgCl2) | align="center" valign="bottom" | 1X | align="center" 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="center" valign="bottom" | 2.5 mM | align="center" align="center" valign="bottom" | 25 | align="center" valign="bottom" | 2.5 mM |- style="font-size:12pt" | height="15" valign="bottom" | 25 mM MgCl2 | align="center" valign="bottom" | 3.5 mM | align="center" align="center" valign="bottom" | 6 | align="center" valign="bottom" | 3.5 mM |- style="background-color:#BFBFBF;font-size:12pt" | height="15" valign="bottom" | 100 uM Primer A (Free primer-Primer 2) | align="center" valign="bottom" | 9 uM | align="center" align="center" valign="bottom" | 5 | align="center" valign="bottom" | 5 uM |- style="font-size:12pt" | height="15" valign="bottom" | 10 uM Primer B (Bound primer-Primer 1) | align="center" valign="bottom" | 0.1 uM | align="center" align="center" valign="bottom" | 1 | align="center" valign="bottom" | 0.1 uM |- style="background-color:#BFBFBF;font-size:12pt" | height="15" valign="bottom" | Hydrogel beads, 2M/uL | align="center" valign="bottom" | 50 Million | align="center" align="center" valign="bottom" | 10 | align="center" valign="bottom" | 60 M |- style="font-size:12pt" | height="15" valign="bottom" | DreamTaq Polymerase (5 U/uL) * | align="center" valign="bottom" | 90 Units | align="center" align="center" valign="bottom" | 18 | align="center" valign="bottom" | 90 Units |- style="background-color:#BFBFBF;font-size:12pt" | height="30" | DNA template 2nd Dilution (Barcode Oligos 1 pM) | align="center" | 0.12 pM | align="center" align="center" | 10 | align="center" valign="bottom" | 0.012 fmol total (0.006 fmol each?) |- style="font-size:12pt" | height="15" valign="bottom" | 1 M Ammonium Sulfate | align="center" valign="bottom" | 5 mM | align="center" align="center" valign="bottom" | 0 | align="center" align="center" valign="bottom" | 0 |- style="background-color:#BFBFBF;font-size:12pt" | height="15" valign="bottom" | 1 mM Spermidine*** | align="center" valign="bottom" | 100 uM | align="center" align="center" valign="bottom" | 10 | align="center" align="center" valign="bottom" | 0 |- style="font-size:12pt" | height="15" valign="bottom" | TIPP | align="center" valign="bottom" | NA | align="center" align="center" valign="bottom" | 0 | align="center" align="center" valign="bottom" | 0 |- style="background-color:#BFBFBF;font-size:12pt" | height="15" valign="bottom" | Water | align="center" valign="bottom" | NA | align="center" align="center" valign="bottom" | 5 | align="center" valign="bottom" | NA |- style="font-size:12pt" | height="15" valign="bottom" | Total Volume | align="center" align="center" valign="bottom" | |style="font-weight:bold" align="center" align="center" valign="bottom" | 100 | align="center" align="center" valign="bottom" | |} <li>Aliquot 90 uL master mix into 4 tubes</li> <li>Add 10 uL appropriate primers according to sample table</li> <li>Aliquot 50 uL each non-emulsion sample into new tubes (A/B)</li> </ol> <li>Oil-Aqueous Mixing</li> <ol type="A"> <li>Mix 20 uL aqueous into 200 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 300 uL total volume into 6 tubes, 50 uL each; use same pipette tip; only made 5 (oil sticks to 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 40 times</li> <li>95C 30 sec</li> <li>68C 3 min</li> <li>Goto f 20X times</li> <li>4C forever</li> </ol></ol> </ol> Continued [[Daniel:Notebook/PosSequencing/2017-5-30|Tuesday, May 30]] =Barcode Oligo Production (Started [[Daniel:Notebook/PosSequencing/2017-5-24|Yesterday]])= ==Protocol== <ol start="2"> <li>Qiaquick Column</li> '''Note''': I will be pooling samples and double loading columns, yielding 4 samples per column (2 columns per barcode oligo, 12 columns total) <ol type="A"> <li>Pool 4 samples and 1000 uL PB (5X binding buffer)</li> <li>Load sample onto column and spin for 1 minute at 14000 rpm; discard flow through</li> <li>Add another 600 uL pooled sample to columnand spin for 1 minute at 14000 rpm; discard flow through <li>Add 700 uL PE (wash buffer) to column and spin for 1 minute at 14000 rpm; discard flow through</li> <li>Dry spin column for 1 minute at 14000 rpm; discard flow through</li> <li>Let stand with cover open in fume hood for ~5 minutes</li> <li>Transfer column to a new 1.5 mL eppendorf tube</li> <li>Add 35 uL nfH<sub>2</sub>O to column</li> <li>Let stand 1 minute</li> <li>Spin for 1 minute at 14000 rpm</li> <li>Pool samples when finished; concentrate if desired</li> </ol> <li>TBE Gel</li> <ol type="A"> <li>Mix 160 uL TBE and 40 uL 6X dye</li> <li>Aliquot 10 uL mix to parafilm</li> <li>Add 2 uL sample or 1.5 uL ladder to appropriate aliquot</li> <li>Add 10 uL mix to appropriate lane</li> <li>Run gel at 240V for 24 minutes</li> <li>Open gel and stain with 2 uL SYBR gold for 3 minutes</li> <li>Rinse and image in gel doc</li> </ol> <li>Qubit</li> <ol type="A"> <li>Mix 1194 uL dsDNA buffer and 6 uL dye</li> <li>Aliquot 190 uL to standard tubes and 199 uL to dye tubes</li> <li>Add 10 uL standard or 1 uL dye</li> <li>Vortex and spin down</li> <li>Let sit 5 minutes in the dark</li> <li>Measure in the Qubit</li> </ol></ol> ==Results== {| class="wikitable" <hiddentext>generated with [[:de:Wikipedia:Helferlein/VBA-Macro for EXCEL tableconversion]] V1.8</hiddentext> |- style="background-color:#66FF66;font-size:12pt;font-weight:bold" align="center" | width="190" height="45" | Sample | width="85" | Qubit Conc (ug/mL) | width="100" | Amount (nM) | width="95" | Total Amount (fmol) | width="95" | Amplification (Over 40 amol) |- style="font-size:12pt" | height="15" valign="bottom" | Barcode Oligo v1 (EcoRI) | align="center" align="center" | 33.0 | align="center" align="center" | 526.3 | align="center" align="center" | 3.16E+04 | align="center" align="center" | 7.89E+08 |- style="background-color:#BFBFBF;font-size:12pt" | height="15" valign="bottom" | Barcode Oligo v2 (EcoRI) | align="center" align="center" | 20.0 | align="center" align="center" | 319.0 | align="center" align="center" | 2.87E+04 | align="center" align="center" | 7.18E+08 |- style="font-size:12pt" | height="15" valign="bottom" | Barcode Oligo v3 (EcoRI) | align="center" align="center" | 33.7 | align="center" align="center" | 537.5 | align="center" align="center" | 3.22E+04 | align="center" align="center" | 8.06E+08 |- style="background-color:#BFBFBF;font-size:12pt" | height="15" valign="bottom" | Barcode Oligo v1 (Uracil) | align="center" align="center" | 26.3 | align="center" align="center" | 419.5 | align="center" align="center" | 2.52E+04 | align="center" align="center" | 6.29E+08 |- style="font-size:12pt" | height="15" valign="bottom" | Barcode Oligo v2 (Uracil) | align="center" align="center" | 32.6 | align="center" align="center" | 519.9 | align="center" align="center" | 3.12E+04 | align="center" align="center" | 7.80E+08 |- style="background-color:#BFBFBF;font-size:12pt" | height="15" valign="bottom" | Barcode Oligo v3 (Uracil) | align="center" align="center" | 33.6 | align="center" align="center" | 535.9 | align="center" align="center" | 3.22E+04 | align="center" align="center" | 8.04E+08 |} <gallery perrow=2 heights=250px widths=250px mode=packed-hover> File:2017-05-25-PosSeq-BarcProduction-EcoRI.png|EcoRI Samples File:2017-05-25-PosSeq-BarcProduction-Uracil.png|Uracil samples </gallery> [[Category:20170524]] [[Category:20170525]] [[Category:OligoPrep]] [[Category:PosSeq]]
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