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=RCA Test (Started [[Daniel:Notebook/ComboLock/2017-3-24|March 24]])= [[Daniel:Notebook/ComboLock|Back to Calendar]] ==Protocol== <ol> <li>Rolling Circle Amplification</li> <ol type="A" start="9"> <li>Add 20 uL nfH2O to every sample</li></ol> <li>qPCR</li> <ol type="A"> <li>Make master mixes according to 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="240" height="30" | Reagent | width="85" | 1X Rxn | width="140" | MM-Amp (33.2X) | width="100" | MM-Prod (9.2X) |- style="font-size:12pt" | height="15" valign="bottom" | Primer Pair | align="center" valign="bottom" | NA | align="center" valign="bottom" | AmpF/AmpR | align="center" valign="bottom" | p4/p12RC |- style="background-color:#BFBFBF;font-size:12pt" | height="15" valign="bottom" | Forward Primer (10 uM) | align="center" align="center" valign="bottom" | 1 | align="center" align="center" valign="bottom" | 33.2 | align="center" align="center" valign="bottom" | 9.2 |- style="font-size:12pt" | height="15" valign="bottom" | Forward Primer (10 uM) | align="center" align="center" valign="bottom" | 1 | align="center" align="center" valign="bottom" | 33.2 | align="center" align="center" valign="bottom" | 9.2 |- style="background-color:#BFBFBF;font-size:12pt" | height="15" valign="bottom" | Kapa SYBR Fast Master Mix (2X) | align="center" align="center" valign="bottom" | 25 | align="center" align="center" valign="bottom" | 830 | align="center" align="center" valign="bottom" | 230 |- style="font-size:12pt" | height="15" valign="bottom" | nfH2O | align="center" align="center" valign="bottom" | 21 | align="center" align="center" valign="bottom" | 697.2 | align="center" align="center" valign="bottom" | 193.2 |- style="background-color:#BFBFBF;font-size:12pt" | height="15" valign="bottom" | Total |style="font-weight:bold" align="center" align="center" valign="bottom" | 48 |style="font-weight:bold" align="center" align="center" valign="bottom" | 1593.6 |style="font-weight:bold" align="center" align="center" valign="bottom" | 441.6 |} <li>Add 48 uL master mix to each well</li> <li>Add 2 uL sample according to plate layout</li> [[Image:PlateLayout-20170327-RCATest-CPLP.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>Qubit Measurement</li> <ol type="A"> <li>Add 995 uL buffer and 5 uL ssdye to tube</li> <li>Add 190 uL buffer and 10 uL correct standard to standard tubes</li> <li>Add 199 uL buffer and 1 uL sample to sample tubes</li> <li>Let sit 5-10 minutes in the dark</li> <li>Measure in Qubit</li></ol> <li>TBE Gel</li> <ol type="A"> <li>Mix 120 uL TBE and 40 uL 6X dye</li> <li>Aliquot 10 uL per sample on to parafilm</li> <li>Add 2 uL sample or 1.5 uL ladder to aliquots</li> <li>Mix and add 10 uL per lane to gel</li> <li>Run gel at 230V for 24 minutes</li> <li>Add 2 uL SYBR gold and incubate on shaker for 3 minutes</li> <li>Rinse and image in gel doc</li></ol> </ol> ==Results== ===Qubit Results=== {| class="wikitable" <hiddentext>generated with [[:de:Wikipedia:Helferlein/VBA-Macro for EXCEL tableconversion]] V1.8</hiddentext> |- style="background-color:#66FFCC;font-size:13pt;font-weight:bold" align="center" | width="120" height="60" | Sample Name | width="250" | Condition | width="85" | ug/mL | width="85" | ng/mL | width="85" | bp | width="85" | nM |- style="font-size:13pt" | height="17" align="center" valign="bottom" | XA | align="center" valign="bottom" | NEB RCA 3 hrs CP (LLRC) | align="center" align="center" valign="bottom" | 4.27 | align="center" valign="bottom" | | align="center" align="center" valign="bottom" | 188 | align="center" align="center" valign="bottom" | 68.8 |- style="background-color:#BFBFBF;font-size:13pt" | height="17" align="center" valign="bottom" | XB | align="center" valign="bottom" | NEB RCA 3 hrs CP (LLRC*) | align="center" align="center" valign="bottom" | 24 | align="center" valign="bottom" | | align="center" align="center" valign="bottom" | 188 | align="center" align="center" valign="bottom" | 386.9 |- style="font-size:13pt" | height="17" align="center" valign="bottom" | XC | align="center" valign="bottom" | NEB RCA 3 hrs CP (LLRC***) | align="center" align="center" valign="bottom" | 37.4 | align="center" valign="bottom" | | align="center" align="center" valign="bottom" | 188 | align="center" align="center" valign="bottom" | 602.8 |- style="background-color:#BFBFBF;font-size:13pt" | height="17" align="center" valign="bottom" | XD | align="center" valign="bottom" | NEB RCA 3 hrs LP (LLRC) | align="center" align="center" valign="bottom" | 2.66 | align="center" valign="bottom" | | align="center" align="center" valign="bottom" | 188 | align="center" align="center" valign="bottom" | 42.9 |- style="font-size:13pt" | height="17" align="center" valign="bottom" | XE | align="center" valign="bottom" | NEB RCA 3 hrs LP (LLRC*) | align="center" align="center" valign="bottom" | 24.9 | align="center" valign="bottom" | | align="center" align="center" valign="bottom" | 188 | align="center" align="center" valign="bottom" | 401.4 |- style="background-color:#BFBFBF;font-size:13pt" | height="17" align="center" valign="bottom" | XF | align="center" valign="bottom" | NEB RCA 3 hrs LP (LLRC***) | align="center" align="center" valign="bottom" | 37.5 | align="center" valign="bottom" | | align="center" align="center" valign="bottom" | 188 | align="center" align="center" valign="bottom" | 604.5 |- style="font-size:13pt" | height="17" align="center" valign="bottom" | CP | align="center" valign="bottom" | Circular Product Alone | align="center" align="center" valign="bottom" | 12.1 | align="center" valign="bottom" | | align="center" align="center" valign="bottom" | 188 | align="center" align="center" valign="bottom" | 195.0 |- style="background-color:#BFBFBF;font-size:13pt" | height="17" align="center" valign="bottom" | PA | align="center" valign="bottom" | NEB RCA 6 hrs CP (LLRC) | align="center" align="center" valign="bottom" | 3.08 | align="center" valign="bottom" | | align="center" align="center" valign="bottom" | 188 | align="center" align="center" valign="bottom" | 49.7 |- style="font-size:13pt" | height="17" align="center" valign="bottom" | PB | align="center" valign="bottom" | NEB RCA 6 hrs CP (LLRC*) | align="center" align="center" valign="bottom" | 17.5 | align="center" valign="bottom" | | align="center" align="center" valign="bottom" | 188 | align="center" align="center" valign="bottom" | 282.1 |- style="background-color:#BFBFBF;font-size:13pt" | height="17" align="center" valign="bottom" | PC | align="center" valign="bottom" | NEB RCA 6 hrs CP (LLRC***) | align="center" align="center" valign="bottom" | 28.3 | align="center" valign="bottom" | | align="center" align="center" valign="bottom" | 188 | align="center" align="center" valign="bottom" | 456.2 |- style="font-size:13pt" | height="17" align="center" valign="bottom" | PD | align="center" valign="bottom" | NEB RCA 6 hrs LP (LLRC) | align="center" align="center" valign="bottom" | 1.84 | align="center" valign="bottom" | | align="center" align="center" valign="bottom" | 188 | align="center" align="center" valign="bottom" | 29.7 |- style="background-color:#BFBFBF;font-size:13pt" | height="17" align="center" valign="bottom" | PE | align="center" valign="bottom" | NEB RCA 6 hrs LP (LLRC*) | align="center" align="center" valign="bottom" | 17.9 | align="center" valign="bottom" | | align="center" align="center" valign="bottom" | 188 | align="center" align="center" valign="bottom" | 288.5 |- style="font-size:13pt" | height="17" align="center" valign="bottom" | PF | align="center" valign="bottom" | NEB RCA 6 hrs LP (LLRC***) | align="center" align="center" valign="bottom" | 29.5 | align="center" valign="bottom" | | align="center" align="center" valign="bottom" | 188 | align="center" align="center" valign="bottom" | 475.5 |- style="background-color:#BFBFBF;font-size:13pt" | height="17" align="center" valign="bottom" | LP | align="center" valign="bottom" | Linear Product Alone | align="center" valign="bottom" | | align="center" align="center" valign="bottom" | 450 | align="center" align="center" valign="bottom" | 188 | align="center" align="center" valign="bottom" | 7.3 |- style="font-size:13pt" | height="17" align="center" valign="bottom" | QA | align="center" valign="bottom" | NEB RCA 12 hrs CP (LLRC) | align="center" align="center" valign="bottom" | 4.87 | align="center" valign="bottom" | | align="center" align="center" valign="bottom" | 188 | align="center" align="center" valign="bottom" | 78.5 |- style="background-color:#BFBFBF;font-size:13pt" | height="17" align="center" valign="bottom" | QB | align="center" valign="bottom" | NEB RCA 12 hrs CP (LLRC*) | align="center" align="center" valign="bottom" | 20.2 | align="center" valign="bottom" | | align="center" align="center" valign="bottom" | 188 | align="center" align="center" valign="bottom" | 325.6 |- style="font-size:13pt" | height="17" align="center" valign="bottom" | QC | align="center" valign="bottom" | NEB RCA 12 hrs CP (LLRC***) | align="center" align="center" valign="bottom" | 29.6 | align="center" valign="bottom" | | align="center" align="center" valign="bottom" | 188 | align="center" align="center" valign="bottom" | 477.1 |- style="background-color:#BFBFBF;font-size:13pt" | height="17" align="center" valign="bottom" | QD | align="center" valign="bottom" | NEB RCA 12 hrs LP (LLRC) | align="center" align="center" valign="bottom" | 1.97 | align="center" valign="bottom" | | align="center" align="center" valign="bottom" | 188 | align="center" align="center" valign="bottom" | 31.8 |- style="font-size:13pt" | height="17" align="center" valign="bottom" | QE | align="center" valign="bottom" | NEB RCA 12 hrs LP (LLRC*) | align="center" align="center" valign="bottom" | 19.2 | align="center" valign="bottom" | | align="center" align="center" valign="bottom" | 188 | align="center" align="center" valign="bottom" | 309.5 |- style="background-color:#BFBFBF;font-size:13pt" | height="17" align="center" valign="bottom" | QF | align="center" valign="bottom" | NEB RCA 12 hrs LP (LLRC***) | align="center" align="center" valign="bottom" | 33.7 | align="center" valign="bottom" | | align="center" align="center" valign="bottom" | 188 | align="center" align="center" valign="bottom" | 543.2 |} ===qPCR Results=== <gallery perrow=3 heights=300px widths=300px> File:20170327-qPCR-RCATest-CPLP-EnzymeComparison.png|qPCR curves showing enzyme comparison (All 3hrs) File:20170327-qPCR-RCATest-CPLP-TimeComparison.png|qPCR curves showing RCA Time comparison (NEB) File:20170327-qPCR-RCATest-CPLP-AverageComparison.png|qPCR curves showing Averaged comparisons (All conditions) File:2017-03-27-RCATest-CPLP-NEB-3hr.png|Gel image-NEB-3hr File:2017-03-27-RCATest-CPLP-TF-3hr.png|Gel image-TF-3hr File:2017-03-27-RCATest-CPLP-Epi-3hr.png|gel image-Epi-3hr File:2017-03-27-RCATest-CPLP-NEB-6hr.png|Gel image-NEB-6hr File:2017-03-27-RCATest-CPLP-NEB-12hr.png|Gel image-NEB-12hr File:2017-03-27-RCATest-CPLP-NEB-adapter.png|Gel image-NEB-3hr-adapters </gallery> ===Summary Results and Discussion=== <gallery perrow=2 heights=300px widths=300px> File:20170327-RCATest-CPLP-CT-EnzymeComparison.png|Comparing the three enzymes (3 hrs RCA) File:20170327-RCATest-CPLP-CT-TimeComparison.png|Comparing CT values between RCA times (NEB) </gallery> Based on the results here, I would say that the Thermo enzyme seemed to be the best (keep in mind Epicentre was mixed 1:10 before adding it to the thermocycler and is therefore not necessarily the best true comparison for Epi). Also, the amount of product does in fact amplify by time accordingly, that is for each different RCA primer (LLRC, LLRC*, and LLRC***) the amount of product present at 6 hours is approximately double the amount at 3 hours, and 12 hours is approximately double 6 hours. However, the baseline still appears to be off. That is, the total amount present should be '''much''' higher than is being elucidated in the qPCR at the moment. [[Category:ComboLock]] [[Category:20170324]]
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