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=Emulsion PCR Test-Dual Biotin (Started [[Daniel:Notebook/PosSequencing/2017-6-14|Yesterday]])= [[Daniel:Notebook/PosSequencing|Back to Calendar]] ==Protocol== <ol start="5"> <li>Prepare Water saturated compounds</li> <ol type="A"> <li>Mix 3 mL diethyl ether and 3 mL ddH2O in a falcon tube; allow to settle, extract top (ether) phase</li> <li>Mix 3 mL ethyl acetate and 3 mL ddH2O in a falcon tube; allow to settle, extract top (acetate) phase</li> </ol> <li>Emulsion Breaking</li> <ol type="A"> <li>With a 200 uL pipette add 100 uL diethyl ether to each sample; save the tip</li> <li>Adjust tip to 200 and using same tip pool samples in a new 1.5 mL tube</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-DE mix (top layer) but do not disrupt the oil/aqueous interface</li> <li>Add 1000 uL ethyl acetate and vortex for 30 seconds</li> <li>Spin down sample at 13,000rpm for 3 minutes</li> <li>Remove ethyl acetate without disturbing the pellet</li> <li>Add 1000 uL diethyl ether and vortex for 30 seconds</li> <li>Spin down sample at 13,000rpm for 3 minutes</li> <li>Remove DE without disturbing the aqueous phase</li> <li>Add 500 uL 4X SSC wash buffer to 1.5 mL tube and vortex for 30 seconds</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 one more time (j to o)</li> <li>On last wash, <span style="background:#FFFF00">take 15 uL (of 100) for qPCR</span></li> </ol> ****** <li>Strand Separation (DNA Denaturation)</li> <ol type="A"> <li>Add 500 uL KOH buffer (pH 12.0)</li> <li>Spin down sample at 13,000rpm for 1 minute</li> <li>Remove all but 40uL buffer</li> <li>Repeat KOH steps A-C once more</li> <li>Add 500 uL 4X SSC buffer (pH 12.0)</li> <li>Spin down sample at 13,000rpm for 1 minute</li> <li>Remove all but 10uL buffer</li> <li>Resuspend in 80 uL 2X SSC buffer</li> <li><span style="background:#FFFF00">Use this for final step of qPCR</span></li> </ol> <li>Single primer qPCR</li> <ol type="A"> <li>Make the following master mixes</li> {| class="wikitable" <hiddentext>generated with [[:de:Wikipedia:Helferlein/VBA-Macro for EXCEL tableconversion]] V1.8</hiddentext> |- style="background-color:#B1A0C7;font-size:12pt;font-weight:bold" align="center" | width="180" height="30" | Reagent | width="95" | uL in Single Rxn | width="95" | Master Mix P2 (4.2X) | width="95" | Master Mix BioU (4.2X) | width="95" | Master Mix Dual (4.2X) |- style="font-size:12pt" | height="15" valign="bottom" | Primer 2 (10 uM) | align="center" align="center" valign="bottom" | 1 | align="center" align="center" valign="bottom" | 4.2 | align="center" align="center" valign="bottom" | 0 | align="center" align="center" valign="bottom" | 4.2 |- style="background-color:#BFBFBF;font-size:12pt" | height="15" valign="bottom" | BioU Primer (10 uM) | align="center" align="center" valign="bottom" | 1 | align="center" align="center" valign="bottom" | 0 | align="center" align="center" valign="bottom" | 4.2 | align="center" align="center" valign="bottom" | 4.2 |- style="font-size:12pt" | height="15" valign="bottom" | 2X Kapa SYBR Master Mix | align="center" align="center" valign="bottom" | 20 | align="center" align="center" valign="bottom" | 84 | align="center" align="center" valign="bottom" | 84 | align="center" align="center" valign="bottom" | 84 |- style="background-color:#BFBFBF;font-size:12pt" | height="15" valign="bottom" | nfH2O | align="center" align="center" valign="bottom" | 14 | align="center" align="center" valign="bottom" | 58.8 | align="center" align="center" valign="bottom" | 58.8 | align="center" align="center" valign="bottom" | 54.6 |- style="font-size:12pt" | height="15" valign="bottom" | Total* (+ 5 uL sample=40) |style="font-weight:bold" align="center" align="center" valign="bottom" | 35 |style="font-weight:bold" align="center" align="center" valign="bottom" | 147 |style="font-weight:bold" align="center" align="center" valign="bottom" | 147 |style="font-weight:bold" align="center" align="center" valign="bottom" | 147 |} <li>Aliquot 35 uL master mix to appropriate lanes</li> <li>Add 2 uL sample according to plate layout below</li> [[Image:PlateLayout-SinglePrimer-DualBiotin-20170615.png|522x348px]] <li>Run the following thermocycler program</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> </ol> ==Results== <gallery perrow=2 heights=250px widths=350px mode=packed-hover> File:PlateLayout-SinglePrimer-DualBiotin-20170615.png|Plate layout File:Singleprimer-20170615-dualbiotin-plateCTs.png|Plate CTs File:Singleprimer-20170615-dualbiotin-plateCTs-normed.png|Plate CTs-normed File:Singleprimer-20170615-dualbiotin-rawcurves.png|Raw curves </gallery> [[Category:PosSeq]] [[Category:20170614]]
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