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=Biotin vs Dual Biotin (Started [[Daniel:Notebook/PosSequencing/2017-6-12|Yesterday]])= [[Daniel:Notebook/PosSequencing|Back to Calendar]] '''Workflow Reminder''' [[Image:ExperimentMap-20170610-Biotin-DualBiotin.png|350x265px]] ==Protocol== <ol start="5"> <li>qPCR</li> <ol type="A"> <li>Prepare 2 plates; 1 single biotin and 1 dual biotin</li> <li>For each sample prepare 58X master mix and (4X) master mix: 1X components set to 30 uL; add 2uL sample</li> <ol type="a"> <li>58 (4) uL 1 uM primer2</li> <li>(4) uL 1 uM biotin/2biotin uracil primer</li> <li>58 uL 10 pM barcode oligo v2</li> <li>870 (60) uL 2X Kapa SYBR</li> <li>638 (44) uL nfH<sub>2</sub>O</li></ol> <li>Add 28 uL master mix according to plate layout (4X master mix is for positive controls, 58X is for samples and negative controls)</li> [[Image:PlateLayout-20170613-BiotinTest.png|350x250px]] <li>Add 2 uL appropriate sample according to plate layout</li> <li>Use "standard" thermocycling program</li> </ol> ==Results== <gallery perrow=2 heights=250px widths=350px mode=packed-hover caption="Single Biotin Results"> File:PlateLayout-20170613-BiotinTest.png|Plate array File:Singlebiotin-20170613-plateCTs.png|Plate CTs File:Singlebiotin-20170613-plateCTs-normed.png|Normalized plate CTs File:Singlebiotin-20170613-rawcurves.png|Raw curves </gallery> <gallery perrow=2 heights=250px widths=350px mode=packed-hover caption="Single Biotin Results"> File:PlateLayout-20170613-BiotinTest.png|Plate array File:Dualbiotin-20170613-plateCTs.png|Plate CTs File:Dualbiotin-20170613-plateCTs-normed.png|Normalized plate CTs File:Dualbiotin-20170613-rawcurves.png|Raw curves </gallery> ==Discussion== I won't bother to do any more complicated analysis than what's above. If you look at the plate CTs the pattern emerges that the dual biotin is far more resilient than the single biotin. At 95C it does experience some losses, but 90C appears to hold well. I can probably get away with short 95C or 90C incubations for doing emPCR. [[Category:PosSeq]] [[Category:20170612]]
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