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=Emulsion PCR= [[Daniel:Notebook/PosSequencing|Back to Calendar]] Not actually run today. Probably [[Daniel:Notebook/PosSequencing/2017-4-26|Wednesday]]. ==Protocol== This protocol is principally based off the Ion Torrent protocol for emulsion PCR <ol> <li>Template DNA Production (Barcode Oligos)</li> <ol type="A"> <li>Start with oligo concentrations of 100 uM (100 pmol/uL)</li> <li>Make dilutions by adding 5 uL into 50 uL (1:10 dilutions)</li> <li>Dilute 8X times (final concentration 1 pM or 1 amol/uL)</li> <li>Make oligo solution by adding 5 uL barcode1 (Alexa488) and barcode3(Cy5) since they have low overlap-Final concentration of oligo is 1 pM; adding 10 uL to PCR mix makes it 0.1pM, the suggested concentration from Ion Torrent protocol</li></ol> <li>Oil Production</li> <ol type="A"> <li>Start from [[Daniel:Notebook/PosSequencing/2017-4-10|Monday April 14]]'s oil prep (Span80-4.5%, Tween20-0.1%, TritonX100-0.05% in mineral oil)</li></ol> <li>Template-Bead Binding</li> <ol type="A"> <li>Suspend 2.5 uL (25 ug) beads in 100 uL Dynabuffer</li> <li>Apply magnet for 30 sec and remove supernatant</li> <li>Suspend 2.5 uL (25 ug) beads in 20 uL Dynabuffer (conc. 1 ug/uL)</li> <li>Add 10 uL 1 uM (10 pmol total, 10X excess) template oligo and 10 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 5 uL TE buffer</li> <li>Mix together the three suspensions of beads with different oligos</li> <li>Prepare the aqueous mix according to the table below</li> <li>Mix ingredients using the following table (see '''Notes''')</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="250" height="34" | Component | width="150" | Ion Torrent Concentration | width="120" | My uL Added | width="140" | My Final Concentration |- style="font-size:12pt" | height="15" valign="bottom" | 5X PCR Buffer (Taq) * | align="center" valign="bottom" | 1X | align="center" align="center" valign="bottom" | 20 | 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" | 14 | 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" | Taq? Polymerase (5 U/uL) * | align="center" valign="bottom" | 90 Units | align="center" align="center" valign="bottom" | 10 | align="center" align="center" valign="bottom" | |- style="background-color:#BFBFBF;font-size:12pt" | height="30" | DNA template 2nd Dilution (Barcode Oligos) | 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" | 0 | 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" valign="bottom" | 100 | align="center" valign="bottom" | |} </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</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>68C 30 sec</li> <li>Goto b 40 times</li> <li>95C 30 sec</li> <li>68C 6 min</li> <li>Goto f 20X times</li> <li>4C forever</li> </ol></ol> ==Protocol Part 2== <ol start="6"> <li>Emulsion Breaking</li> <ol type="A"> <li>With a 200 uL pipette pool the emulsion into a 1.5 mL tube; save the tip and put aside</li> <li>Add 100 uL isopropyl alcohol (IPA) to tube with 200 uL tip (use new tip)</li> <li>Draw up IPA with tip from part A, mix to resuspend tip</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-IPA mix (top layer) but do not disrupt the oil/aqueous interface</li> <li>Add 1000 uL IPA and vortex for 30 seconds</li> <li>Spin down sample at 13,000rpm for 3 minutes</li> <li>Again remove IPA without disturbing the pellet</li> <li>Add 500 uL 4X SSC wash buffer to 1.5 mL tube and vortex for 30 seconds</li> <li>Go back and mix thoroughly by pipetting up and down; the pellet may be hard to resuspend</li> <li>Leave on 1.5mL mixer at 1300rpm 1 minute</li> <li>Spin down sample at 13,000rpm for 1 minute</li> <li>Remove all but 40uL wash buffer</li> <li>Repeat steps 11 to 15 two times (j to o)</li> <li>Resuspend in ~100 uL 4X SSC buffer</li></ol> <li>Fluorescent Oligo Binding</li> <li></li> <li>Imaging</li> </ol> ==Notes== * *: NEB Taq has terrible information. Since the ThermoFisher Taq is much better documented and available at a reasonable price, I will go with that in the future. It is on the order sheet and I will switch to that in the future. Since I'm not running the protocol today (It's almost 5), the Thermo * **: dNTPs are also available at up to 100 mM for each dNTP if this concentration proves too annoying to work with</li> * ***: Spermidine seems useful so I'm ordering it. We may actually have Ammonium sulfate but I'm not sure it's necessary</li>
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