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=Bead qPCR Test= [[Daniel:Notebook/PosSequencing|Back to Calendar]] This experiment has 2 purposes: to determine the density of beads necessary to optically block qPCR detection (and hopefully some levels that don't) as well as the efficiency of bead-based qPCR vs aqueous. This will hopefully help me make the next experiment run smoother, specifically using [https://tools.thermofisher.com/content/sfs/manuals/MAN0007720.pdf digital qPCR] to amplify the beads. ==Protocol== <ol> <li>Template-Bead Binding; Make 4 samples and follow this protocol for each</li> <ol type="A"> <li>Suspend 5 uL (50 ug, ~50M 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> <li>Wash twice with 100 uL Dynabuffer</li> <li>Resuspend beads in 10 uL TE buffer</li> </ol> <li>Dilutions</li> <ol type="A"> <li>Make dilutions using 2 uL suspended beads with 18 uL TE for dilutions</li> <li>Make dilutions of 1:10, 1:100, 1:1000, and 1:10000</li> '''Sample Matrix''' {| 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="59" height="40" | Beads in Sample | width="65" | Dilution (Power 10) | width="65" | Amount Added (uL) | width="85" | ~Amt BioU Primer (pmol) |- style="font-size:12pt" | height="15" valign="bottom" | 10 M | align="right" align="center" valign="bottom" | 0 | align="right" align="center" valign="bottom" | 2 | align="center" valign="bottom" | 5 pmol |- style="background-color:#BFBFBF;font-size:12pt" | height="15" valign="bottom" | 2 M | align="right" align="center" valign="bottom" | 1 | align="right" align="center" valign="bottom" | 4 | align="center" valign="bottom" | 1 pmol |- style="font-size:12pt" | height="15" valign="bottom" | 1 M | align="right" align="center" valign="bottom" | 1 | align="right" align="center" valign="bottom" | 2 | align="center" valign="bottom" | 500 nmol |- style="background-color:#BFBFBF;font-size:12pt" | height="15" valign="bottom" | 0.2 M | align="right" align="center" valign="bottom" | 2 | align="right" align="center" valign="bottom" | 4 | align="center" valign="bottom" | 100 nmol |- style="font-size:12pt" | height="15" valign="bottom" | 0.1 M | align="right" align="center" valign="bottom" | 2 | align="right" align="center" valign="bottom" | 2 | align="center" valign="bottom" | 50 nmol |- style="background-color:#BFBFBF;font-size:12pt" | height="15" valign="bottom" | 20 K | align="right" align="center" valign="bottom" | 3 | align="right" align="center" valign="bottom" | 4 | align="center" valign="bottom" | 10 nmol |- style="font-size:12pt" | height="15" valign="bottom" | 10 K | align="right" align="center" valign="bottom" | 3 | align="right" align="center" valign="bottom" | 2 | align="center" valign="bottom" | 5 nmol |- style="background-color:#BFBFBF;font-size:12pt" | height="15" valign="bottom" | 1 K | align="right" align="center" valign="bottom" | 4 | align="right" align="center" valign="bottom" | 2 | align="center" valign="bottom" | 1 nmol |} </ol> <li>qPCR</li> <ol type="A"> <li>Make the following master mix</li> {| class="wikitable" <hiddentext>generated with [[:de:Wikipedia:Helferlein/VBA-Macro for EXCEL tableconversion]] V1.8</hiddentext> |- style="background-color:#95B3D7;font-size:12pt;font-weight:bold" align="center" | width="180" height="40" | Reagent | width="95" | 1X Added | width="95" | Master Mix (21.2X) |- style="font-size:12pt" | height="15" valign="bottom" | Primer 2 (10 uM) | align="right" align="center" valign="bottom" | 1 | align="right" align="center" valign="bottom" | 21.2 |- style="background-color:#BFBFBF;font-size:12pt" | height="15" valign="bottom" | Barcode Oligo 100 pM | align="right" align="center" valign="bottom" | 1 | align="right" align="center" valign="bottom" | 21.2 (1) |- style="font-size:12pt" | height="15" valign="bottom" | Beads | align="right" align="center" valign="bottom" | 4 | align="right" align="center" valign="bottom" | 84.8 |- style="background-color:#BFBFBF;font-size:12pt" | height="15" valign="bottom" | Kapa 2X SYBR Fast Mix | align="right" align="center" valign="bottom" | 20 | align="right" align="center" valign="bottom" | 424 |- style="font-size:12pt" | height="15" valign="bottom" | nfH2O | align="right" align="center" valign="bottom" | 14 | align="right" align="center" valign="bottom" | 296.8 (155.4) |- style="background-color:#BFBFBF;font-size:12pt" | height="15" valign="bottom" | Total | align="right" align="center" valign="bottom" | 40 | align="right" align="center" valign="bottom" | 848 |} <li>Add 36 uL master mix to each lane</li> <li>Add n uL of sample and 4-n uL nfH<sub>2</sub>O to appropriate lanes according to plate layout</li> <li>For the "Supernatant" sample add 10 uL TE to original beads (0 dilution), mix to suspend, then pull down; take 4 uL supernatant for sample</li> [[Image:PlateLayout-BeadsqPCR-20170621.png|350x250px]] <li>Vortex and spin down after sealing wells</li> <li>Use the [[Daniel:Protocols/qPCR#Presets|standard]] thermocycler program->40 cycles</li> </ol></ol> ==Results== <gallery perrow=2 heights=250px widths=300px mode=packed-hover> File:PlateLayout-BeadsqPCR-20170621.png|Plate layout File:BeadqPCR-plateCTs-normed.png|Plate CTs File:BeadqPCR-rawcurves.png|Raw curves File:BeadqPCRTest-20170621.png|Cycle threshold vs. input (log pmol) </gallery> ==Discussion== First I'll establish the math behind this. The equations are numbered for convenience. [[Image:Equations-20170622.png]] The important equations are '''3''', the idealized y=mx+b with y as number of cycles and x as log(input), and '''4''', which is '''3''' but corrected for the efficiency (e<sub>x</sub>) of reaction explicitly added. Note that efficiency should be between 0 and 1. So using the above equations for sample and control cycling as a function of input. From those suppositions and using the ''controls'', we can calculate the b' constant as 15.737 and m' as 11.138. From those, we can estimate the constants and efficiencies from the ''samples'' trendline: [[Image:Equations2-20170622.png]] So the '''m''' based calculations doesn't make any sense but '''b''' gives an answer of 0.497, which is reasonable. So maybe it's 50% efficient? If so, the following table will dictate how many cycles it would take to amplify given ammounts. Bolded samples are included but from data. {| class="wikitable" <hiddentext>generated with [[:de:Wikipedia:Helferlein/VBA-Macro for EXCEL tableconversion]] V1.8</hiddentext> |- style="background-color:#92CDDC;font-size:12pt;font-weight:bold" align="center" | width="100" height="28" | Input | width="110" | Cycles |- style="font-size:12pt" align="center" valign="bottom" | height="15" | '''1 pmol''' | align="center" | '''92.5''' |- style="background-color:#BFBFBF;font-size:12pt" align="center" valign="bottom" | height="15" | '''500 nmol''' | align="center" | '''87.5''' |- style="font-size:12pt" align="center" valign="bottom" | height="15" | '''100 nmol''' | align="center" | '''111''' |- style="background-color:#BFBFBF;font-size:12pt" align="center" valign="bottom" | height="15" | 10 nmol | align="center" | 114.0 |- style="font-size:12pt" align="center" valign="bottom" | height="15" | 1 nmol | align="center" | 119.7 |- style="background-color:#BFBFBF;font-size:12pt" align="center" valign="bottom" | height="15" | 100 fmol | align="center" | 125.4 |- style="font-size:12pt" align="center" valign="bottom" | height="15" | 10 fmol | align="center" | 131.1 |- style="background-color:#BFBFBF;font-size:12pt" align="center" valign="bottom" | height="15" | 1 fmol | align="center" | 136.8 |- style="font-size:12pt" align="center" valign="bottom" | height="15" | 100 amol | align="center" | 142.5 |} [[Category:PosSeq]] [[Category:20170621]]
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