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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]]
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