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=RCA + Circularization Test (Started [[Daniel:Notebook/ComboLock/2017-2-27|Yesterday]])= [[Daniel:Notebook/ComboLock|Back to Calendar]] ==Protocol-Part 3== <ol start="7"> <li>ssQubit</li> <ol type="A"> <li>Mix together 796 uL Qubit ssDNA buffer and 4 uL dye (200:1 ratio)</li> <li>Add 190 uL mix and 10 uL S1 or S2 to standard tubes</li> <li>Add 199 uL mix and 1 uL sample to sample tubes</li> <li>Incubate 5 minutes in the dark</li> <li>Measure in Qubit; use new run and calibrate with standards</li> <li>'''Results''': Both too low to mean anything.</li></ol> <li>Column Purification-[[Daniel:Protocols/Qiaquick Column|Qiaquick]]</li> <ol type="A"> <li>Add 250 uL (5X) PB (binding buffer) to sample</li> <li>Load sample onto column and spin for 1 minute at 14000 rpm; discard flow through</li> <li>Add 750 uL PE (wash buffer) to column and spin for 1 minute at 14000 rpm; discard flow through;</li> <li>Repeat wash step;</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>Elute with 40 uL EB</li> <li>Let stand 1 minute</li> <li>Spin for 1 minute at 14000 rpm</li></ol> <li>Rolling Circle Amplification</li> <ol type="A"> <li>Prepare master mixes with following enzyme layout following the table in this section</li> {| class="wikitable" <hiddentext>generated with [[:de:Wikipedia:Helferlein/VBA-Macro for EXCEL tableconversion]] V1.8</hiddentext> |- style="background-color:#C4D79B;font-size:12pt;font-weight:bold" align="center" | width="180" height="30" | Samples | width="200" | Condition |- style="font-size:12pt" | height="15" valign="bottom" | Sample X (1-2,ABC) | align="center" valign="bottom" | NEB |- style="background-color:#BFBFBF;font-size:12pt" | height="15" valign="bottom" | Sample Y (1-2,ABC) | align="center" valign="bottom" | Thermo Fisher |- style="font-size:12pt" | height="15" valign="bottom" | Sample Z (1-2,ABC) | align="center" valign="bottom" | Epicenter |- style="background-color:#BFBFBF;font-size:12pt" | height="15" valign="bottom" | A Samples (1-2, XYZ) | align="center" valign="bottom" | Normal Primer |- style="font-size:12pt" | height="15" valign="bottom" | B Samples (1-2, XYZ) | align="center" valign="bottom" | Phosphorothiorate Primer |- style="background-color:#BFBFBF;font-size:12pt" | height="15" valign="bottom" | C Samples (1-2, XYZ) | align="center" valign="bottom" | Column Purified Product |} <li>Add 11.5 uL master mix to each tube</li> <li>Add 2.5 uL LLRC-01 (10 uM) primer to A and C samples; add 2.5 uL LLRC* (10 uM) primer to B samples</li> <li>Add 5 uL sample to new reaction tubes according to following table</li> {| class="wikitable" <hiddentext>generated with [[:de:Wikipedia:Helferlein/VBA-Macro for EXCEL tableconversion]] V1.8</hiddentext> |- style="background-color:#8DB4E2;font-size:12pt;font-weight:bold" align="center" | width="180" height="35" | Reagent | width="80" | Single Rxn Vol | width="85" | MM-X (NEB) uL | width="85" | MM-Y (TF) uL | width="85" | MM-Z (Epi) uL |- style="font-size:12pt" | height="15" valign="bottom" | Template | align="right" align="center" valign="bottom" | 5 | align="right" align="center" valign="bottom" | 0 | align="right" align="center" valign="bottom" | 0 | align="right" align="center" valign="bottom" | 0 |- style="background-color:#BFBFBF;font-size:12pt" | height="15" valign="bottom" | RCA Primer (10 uM) | align="right" align="center" valign="bottom" | 2.5 | align="right" align="center" valign="bottom" | 15.25 | align="right" align="center" valign="bottom" | 15.25 | align="right" align="center" valign="bottom" | 15.25 |- style="font-size:12pt" | height="15" valign="bottom" | dNTP (1 mM) | align="right" align="center" valign="bottom" | 5 | align="right" align="center" valign="bottom" | 30.5 | align="right" align="center" valign="bottom" | 30.5 | align="right" align="center" valign="bottom" | 30.5 |- style="background-color:#BFBFBF;font-size:12pt" | height="15" valign="bottom" | 10X Buffer | align="right" align="center" valign="bottom" | 2 | align="right" align="center" valign="bottom" | 12.2 | align="right" align="center" valign="bottom" | 12.2 | align="right" align="center" valign="bottom" | 12.2 |- style="font-size:12pt" | height="15" valign="bottom" | Phi29 | align="right" align="center" valign="bottom" | 1 | align="right" align="center" valign="bottom" | 0 | align="right" align="center" valign="bottom" | 0 | align="right" align="center" valign="bottom" | 0 |- style="background-color:#BFBFBF;font-size:12pt" | height="15" valign="bottom" | BSA (10 mg/mL) | align="right" align="center" valign="bottom" | 0.4 | align="right" align="center" valign="bottom" | 2.44 | align="right" align="center" valign="bottom" | 0 | align="right" align="center" valign="bottom" | 0 |- style="font-size:12pt" | height="15" valign="bottom" | nfH2O | align="right" align="center" valign="bottom" | 4.1 | align="right" align="center" valign="bottom" | 25.01 | align="right" align="center" valign="bottom" | 27.45 | align="right" align="center" valign="bottom" | 27.45 |- style="background-color:#BFBFBF;font-size:12pt" | height="15" valign="bottom" | Total | align="right" align="center" valign="bottom" | 20 | align="right" align="center" valign="bottom" | 85.4 | align="right" align="center" valign="bottom" | 85.4 | align="right" align="center" valign="bottom" | 85.4 |} <li>Incubate for 5 minutes at 75C to denature sticky strands</li> <li>Lower temperature and incubate for 30 minutes at 55C</li> <li>Lower temperature to 30C; allow at least 1 minute of 30C incubation</li> <li>Add 1 uL correct Phi29 polymerase and incubate 3 hours at 30C</li> <li>Heat kill enzyme with 10 minutes at 65C</li> </ol></ol> ==Protocol-Part 4== <ol start="10"> <li>qPCR</li> <ol type="A"> <li>Make 25.2X qPCR master mix according to following recipe</li> <ol type="a"> <li>529.2 uL nfH2O</li> <li>630 uL SYBR Fast</li> <li>25.2 uL 10 uM AmpF</li> <li>25.2 uL 10 uM AmpR-Ind20</li> </ol> <li>Add 48 uL master mix to each well</li> <li>Add 2 uL sample according to PCR Plate Layout</li> [[Image:PlateLayout-20170228-RCATest.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 x40</li> <li>72C 2 min</li> <li>16C hold</li> </ol></ol> <li>TBE Gel</li> <ol type="A"> <li>Mix 240 uL TBE, 60 uL 6x loading dye</li> <li>Aliquot 10 uL per sample/ladder lane onto parafilm</li> <li>Add 2 uL of sample or ladder to correct drop</li> <li>Load 10 uL in to well</li> <li>Run gel for 23 minutes at 230V</li> <li>Open gel and stain with 3 uL SYBR Gold for 3 minutes</li> <li>Rinse gel and image in gel doc</li> </ol></ol> ===Results=== <gallery perrow=2 heights=300px widths=300px> File:20170228-QPCR-RCATest.png|qPCR curves File:2017-02-28-RCATest-NEB.png|Gel image-NEB RCA File:2017-02-28-RCATest-Gel1.png|Gel image 1 (PreRCA+TF Sample) File:2017-02-28-RCATest-Gel2.png|Gel image 2 (Epicentre +TF No PO4) </gallery> [[Category:ComboLock]] [[Category:20170227]]
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