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=Emulsion PCR Test-Dual Biotin= [[Daniel:Notebook/PosSequencing|Back to Calendar]] I'm actually going to change several things. I know its a big science jump, but we'll call this square 1. Here's the list of alterations: <ol> <li>Dual biotin primers instead of single biotin (duh)</li> <li>'''Oil phase''': I'll make the oil phase according to the [[Daniel:Protocols/EmulsionPCR|Church lab protocol]], which is 4:1 Tegosoft DEC: Mineral Oil. Andrew also recommends the Tegosoft</li> <li>'''Detergent''': Actually keep this one the same: 4.5% Span, 0.4% Tween-80, and 0.05% TritonX-100. This is a mix we have on hand, although I could always order the ABIL-90</li> <li>'''Emulsion Breaking''':I'll use ether instead of IPA. Andrew bought some yesterday and it's supposed to be better</li> <li>'''PCR Protocol''': 94C denaturation steps (hopefully fine with dual biotin), skip second steps of Ion torrent protocol, and cycle 60 times. This is based as much on my [[Daniel:Notebook/PosSequencing/2017-6-13|empirical evidence]] as anything else.</li> <li>'''Taq Concentration''':Pretty all over the board here, but I'll report in terms of Units/uL for comparison. It's really a pretty big spread, but I'll use 2 uL (10U) DreamTaq for a 100 uL reaction (0.1 U/uL, kinda between Murgha and Church/Ion)</li> <li>'''Template Concentration''': Same thing as ''Taq'', better to look at everyone else's versions. Since the Ion Torrent (the one I've been using) is 100X less than I've been using, I'll try with the higher concentrations. Amplifying with more is fine in first trial.</li> </ol> '''Protocol Comparison Table''' {| class="wikitable" <hiddentext>generated with [[:de:Wikipedia:Helferlein/VBA-Macro for EXCEL tableconversion]] V1.8</hiddentext> |- style="background-color:#9BBB59;font-size:12pt;font-weight:bold" | align="center" width="95" height="31" valign="bottom" | | width="95" align="center" | Template | width="100" align="center" | Primers | width="95" align="center" | dNTP | width="140" align="center" | Taq |- style="font-size:12pt" | height="15" valign="bottom" | Church | align="center" valign="bottom" | ???? | align="center" valign="bottom" | 12.5 uM | align="center" valign="bottom" | 3.5 mM | align="center" valign="bottom" | 0.28 U/uL |- style="background-color:#BFBFBF;font-size:12pt" | height="15" valign="bottom" | Shao | align="center" valign="bottom" | 10 pM | align="center" valign="bottom" | 0.4 uM | align="center" valign="bottom" | 0.4 mM | align="center" valign="bottom" | 0.125E-6 U/uL |- style="font-size:12pt" | height="15" valign="bottom" | Murgha | align="center" valign="bottom" | 25 pM | align="center" valign="bottom" | 0.5 uM | align="center" valign="bottom" | 0.2 mM | align="center" valign="bottom" | 0.04 U/uL |- style="background-color:#BFBFBF;font-size:12pt" | height="15" valign="bottom" | Ion Torrent | align="center" valign="bottom" | 0.12 pM | align="center" valign="bottom" | 9 uM | align="center" valign="bottom" | 2.5 mM | align="center" valign="bottom" | 0.6 U/uL |- style="font-size:12pt" | height="15" valign="bottom" | Today's Protocol | align="center" valign="bottom" | 5 pM | align="center" valign="bottom" | 1 uM, 0.1 uM | align="center" valign="bottom" | 1 mM | align="center" valign="bottom" | 0.1 U/uL |} ==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 (50 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="300" height="30" | Component | width="120" | uL Added | width="140" | 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="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="center" valign="bottom" | 1 mM |- style="font-size:12pt" | height="15" valign="bottom" | 25 mM MgCl2 | 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="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="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="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="center" valign="bottom" | 10 Units |- style="background-color:#BFBFBF;font-size:12pt" | height="15" | Barcode Oligo v1 (template, 100 pM) | align="right" align="center" | 5 | align="center" valign="bottom" | 5 pM |- style="font-size:12pt" | height="15" valign="bottom" | 100 mM Ammonium Sulfate | 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="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 |- style="background-color:#BFBFBF;font-size:12pt" | height="15" valign="bottom" | Water | 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 | 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 80 times</li> <li>4C forever</li> </ol></ol> </ol> Continued [[Daniel:Notebook/PosSequencing/2017-6-15|tomorrow]] [[Category:PosSeq]] [[Category:20170614]]
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