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=Probe Production= [[Daniel:Notebook/PosSequencing|Back to Calendar]] Dr Zhang had a good suggestion to do biotin probe production for each of the barcode oligos. I'll do this as a standard reaction without the bead oligos. I can then do the biotin attachment and oligo melting after that. This will allow me to separate the imaging steps from the actual emulsion PCR which will be more important for ==Protocol== <ol> <li>qPCR</li> <ol type="A"> <li>Make 4.2X (1X) master mix with the following reagents</li> <ol type="a"> <li>4.2 (1) uL 10 uM AP1v4U</li> <li>4.2 (1) uL 10 uM BiotinU</li> <li>75.6 (18) uL nfH<sub>2</sub>O</li> <li>105 (25) uL 2X SYBR Fast</li></ol> <li>Add 45 uL master mix to each sample</li> <li>Add 5 uL barcode oligo according to plate layout</li> [[Image:20170505-PlateLayout-BiotinOligoProduction.png|600px]] <li>qPCR Cycles</li> <ol type="a"> <li>95C 3 min</li> <li>95C 3 sec</li> <li>55C 30 sec</li> <li>72C 20 sec</li> <li>plate read</li> <li>goto b x30</li> <li>72C 2 min</li> <li>16C hold</li> </ol></ol> <li>Qiaquick Column</li> <ol type="A"> <li>Add 250 PB (5X binding buffer) to 50 sample</li> <li>Load sample onto column and spin for 1 minute at 14000 rpm; discard flow through</li> <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> </ol> <li>TBE Gel</li> <ol type="A"> <li>Mix 48 uL TBE and 12 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="160" height="37" | Sample | width="65" | Qubit Conc (ug/mL) | width="65" | Amount (nM) | width="101" | Total Amount (fmol) | width="81" | Amplification (Over 5 amol) |- style="font-size:12pt" | height="15" valign="bottom" | Barcode Oligo v1 | align="center" align="center" valign="bottom" | 7.56 | align="center" align="center" valign="bottom" | 238.6 | align="center" align="center" valign="bottom" | 8352.3 | align="center" align="center" valign="bottom" | 1670460 |- style="background-color:#BFBFBF;font-size:12pt" | height="15" valign="bottom" | Barcode Oligo v2 | align="center" align="center" valign="bottom" | 4.85 | align="center" align="center" valign="bottom" | 153.1 | align="center" align="center" valign="bottom" | 5358.3 | align="center" align="center" valign="bottom" | 1071660 |- style="font-size:12pt" | height="15" valign="bottom" | Barcode Oligo v3 | align="center" align="center" valign="bottom" | 6.94 | align="center" align="center" valign="bottom" | 219.1 | align="center" align="center" valign="bottom" | 7667.3 | align="center" align="center" valign="bottom" | 1533460 |- style="background-color:#BFBFBF;font-size:12pt" | height="15" valign="bottom" | NTC | align="center" align="center" valign="bottom" | 0.12 | align="center" align="center" valign="bottom" | 3.8 | align="center" align="center" valign="bottom" | 132.6 | align="center" align="center" valign="bottom" | 26520 |} <gallery perrow=2 heights=300px widths=300px</li> File:20170505-qPCR-BarcodeOligoProduction.png|qPCR curves</li> File:2017-05-05-BarcodeOligoProduction.png|Gel image </gallery> [[Category:PosSeq]] [[Category:20170505]] [[Category:OligoPrep]]
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