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=RCA Test (Started [[Daniel:Notebook/ComboLock/2017-3-24|March 24]])= [[Daniel:Notebook/ComboLock|Back to Calendar]] ==Linear Product Production-Bglii (Started [[Daniel:Notebook/ComboLock/2017-3-25|Yesterday]])== <ol start="2"> <li>TBE gel</li> <ol type="A"> <li>Mix 64 uL TBE and 16 uL 6X dye</li> <li>Aliquot 10 uL on to parafilm per sample/ladder</li> <li>Add 2 uL reaction, 1 uL (10 uM) controls, and 1.5 uL ladder to appropriate aliquot</li> [[Image:GelLanes-20170325-LinearProduct.png|400px]] <li>Add 10 uL mix to gel lanes</li> <li>Run gel for 24 minutes at 230V</li> <li>Stain with 2 uL SYBR gold for 3 minutes</li> <li>Rinse and image in gel doc</li> </ol></ol> [[Image:2017-03-26-LinearProductProduction-Bglii.png|400px]] That's the right one. However, there is a ton of primer (bottom of the gel), so I'll run a ssDNA column to fix. <ol start="3"> <li>ssDNA column</li> <ol type="A"> <li>Add 50 uL '''Binding Buffer''' to the sample; mix well</li> <li>Transfer to '''IIC Column''' and centrifuge at 14000 rpm for 1 minute; '''SAVE THE FLOW THROUGH'''</li> <li>Add 70 uL 100% EtOH to flow through; mix well</li> <li>Transfer to '''IC Column''' and centrifuge at 14000 rpm for 1 minute; discard flow through</li> <li>Add 400 uL '''Prep Buffer''' and centrifuge at 14000 rpm for 1 minute; discard flow through</li> <li>Add 700 uL '''Wash Buffer''' and centrifuge at 14000 rpm for 1 minute; discard flow through</li> <li>Add 400 uL '''Wash Buffer''' and centrifuge at 14000 rpm for 1 minute; discard flow through</li> <li>Centrifuge empty column at 14000 rpm for 2 minutes</li> <li>Transfer to empty 1.5mL centrifuge tube (low bind)</li> <li>Add 20 uL nfH2O and centrifuge at 14000 rpm for 1 minute</li> </ol> ==RCA Protocol== <ol> <li>Rolling Circle Amplification</li> <ol type="A"> <li>Prepare master mix according to table below</li> {| 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="200" height="30" | Reagent | width="85" | Single Rxn (uL) | width="85" | Master Mix X+ (9.2X) | width="85" | Master Mix X- (9.2X) | width="85" | Master Mix Y+ (3.2X) | width="85" | Master Mix Y- (3.2X) | width="85" | Master Mix Z+ (3.2X) | width="85" | Master Mix Z- (3.2X) |- style="font-size:12pt" | height="15" | Enzyme | align="center" | NA | align="center" | NEB | align="center" | NEB | align="center" | Thermo | align="center" | Thermo | align="center" | Epi (1:10) | align="center" | Epi (1:10) |- style="background-color:#BFBFBF;font-size:12pt" | height="15" valign="bottom" | Circular Product (1X) | align="center" align="center" valign="bottom" | 2 | align="center" align="center" valign="bottom" | 18.4 | align="center" align="center" valign="bottom" | 0 | align="center" align="center" valign="bottom" | 6.4 | align="center" align="center" valign="bottom" | 0 | align="center" align="center" valign="bottom" | 6.4 | align="center" align="center" valign="bottom" | 0 |- style="font-size:12pt" | height="15" valign="bottom" | Linear Product (1X) | align="center" align="center" valign="bottom" | 2 | align="center" align="center" valign="bottom" | 0 | align="center" align="center" valign="bottom" | 18.4 | align="center" align="center" valign="bottom" | 0 | align="center" align="center" valign="bottom" | 6.4 | align="center" align="center" valign="bottom" | 0 | align="center" align="center" valign="bottom" | 6.4 |- style="background-color:#BFBFBF;font-size:12pt" | height="15" valign="bottom" | RCA Primer (100 uM) | align="center" align="center" valign="bottom" | 2.5 | align="center" align="center" valign="bottom" | 23 | align="center" align="center" valign="bottom" | 23 | align="center" align="center" valign="bottom" | 8 | align="center" align="center" valign="bottom" | 8 | align="center" align="center" valign="bottom" | 8 | align="center" align="center" valign="bottom" | 8 |- style="font-size:12pt" | height="15" valign="bottom" | dNTP (1 mM) | align="center" align="center" valign="bottom" | 5 | align="center" align="center" valign="bottom" | 46 | align="center" align="center" valign="bottom" | 46 | align="center" align="center" valign="bottom" | 16 | align="center" align="center" valign="bottom" | 16 | align="center" align="center" valign="bottom" | 16 | align="center" align="center" valign="bottom" | 16 |- style="background-color:#BFBFBF;font-size:12pt" | height="15" valign="bottom" | 10X Buffer | align="center" align="center" valign="bottom" | 2 | align="center" align="center" valign="bottom" | 18.4 | align="center" align="center" valign="bottom" | 18.4 | align="center" align="center" valign="bottom" | 6.4 | align="center" align="center" valign="bottom" | 6.4 | align="center" align="center" valign="bottom" | 6.4 | align="center" align="center" valign="bottom" | 6.4 |- style="font-size:12pt" | height="15" valign="bottom" | Phi29 | align="center" align="center" valign="bottom" | 1 | align="center" align="center" valign="bottom" | 0 | align="center" align="center" valign="bottom" | 0 | align="center" align="center" valign="bottom" | 0 | align="center" align="center" valign="bottom" | 0 | align="center" align="center" valign="bottom" | 0 | align="center" align="center" valign="bottom" | 0 |- style="background-color:#BFBFBF;font-size:12pt" | height="15" valign="bottom" | BSA (10 mg/mL) | align="center" align="center" valign="bottom" | 0.4 | align="center" align="center" valign="bottom" | 3.68 | align="center" align="center" valign="bottom" | 3.68 | align="center" align="center" valign="bottom" | 0 | align="center" align="center" valign="bottom" | 0 | align="center" align="center" valign="bottom" | 0 | align="center" align="center" valign="bottom" | 0 |- style="font-size:12pt" | height="15" valign="bottom" | nfH2O | align="center" align="center" valign="bottom" | 8.1 | align="center" align="center" valign="bottom" | 65.32 | align="center" align="center" valign="bottom" | 65.32 | align="center" align="center" valign="bottom" | 24 | align="center" align="center" valign="bottom" | 24 | align="center" align="center" valign="bottom" | 24 | align="center" align="center" valign="bottom" | 24 |- style="background-color:#BFBFBF;font-size:12pt" | height="15" valign="bottom" | Total |style="font-weight:bold" align="center" align="center" valign="bottom" | 20 |style="font-weight:bold" align="center" align="center" valign="bottom" | 117.8 |style="font-weight:bold" align="center" align="center" valign="bottom" | 117.8 |style="font-weight:bold" align="center" align="center" valign="bottom" | 59.52 |style="font-weight:bold" align="center" align="center" valign="bottom" | 59.52 |style="font-weight:bold" align="center" align="center" valign="bottom" | 59.52 |style="font-weight:bold" align="center" align="center" valign="bottom" | 59.52 |} <li>Add 16.5 uL master mix to each tube</li> <ol type="a"><li>Samples "A-C" get + master mixes; samples "D-F" get - master mixes</li></ol> <li>Add 2.5 uL 10 uM RCA primer and to new reaction tubes according to following rules</li> <ol type="a"> <li>"A,D" samples get LLRC</li> <li>"B,E" samples get LLRC-v03 (Mono-phosphorothiorate)</li> <li>"C,F" samples get LLRC-v04 (Tri-phosphorothiorate)</li></ol> <li>Heat reactions up to 95C for 5 min</li> <li>Cool to 55C and incubate for 15 minutes</li> <li>Cool to 30C and add enzyme</li> <li>Incubate at 30C for length of time according to table below</li> {| class="wikitable" <hiddentext>generated with [[:de:Wikipedia:Helferlein/VBA-Macro for EXCEL tableconversion]] V1.8</hiddentext> |- style="background-color:#CCFF66;font-size:12pt;font-weight:bold" align="center" | width="105" height="30" | Sample | width="105" | Master Mix | width="100" | Enzyme | width="100" | Incubation Time (hrs) |- style="font-size:12pt" | height="15" valign="bottom" | X (A-F) | align="center" valign="bottom" | X | align="center" valign="bottom" | NEB | align="center" align="center" valign="bottom" | 3 |- style="background-color:#BFBFBF;font-size:12pt" | height="15" valign="bottom" | Y (A-F) | align="center" valign="bottom" | Y | align="center" valign="bottom" | Thermo | align="center" align="center" valign="bottom" | 3 |- style="font-size:12pt" | height="15" valign="bottom" | Z (A-F) | align="center" valign="bottom" | Z | align="center" valign="bottom" | Epi (1:10) | align="center" align="center" valign="bottom" | 3 |- style="background-color:#BFBFBF;font-size:12pt" | height="15" valign="bottom" | P (A-F) | align="center" valign="bottom" | X | align="center" valign="bottom" | NEB | align="center" align="center" valign="bottom" | 6 |- style="font-size:12pt" | height="15" valign="bottom" | Q (A-F) | align="center" valign="bottom" | X | align="center" valign="bottom" | NEB | align="center" align="center" valign="bottom" | 12 |} <li>Incubate at 65C for 10 minutes;Hold at 10C until next step</li></ol> Continued [[Daniel:Notebook/ComboLock/2017-3-27|tomorrow]] [[Category:ComboLock]] [[Category:20170322]] [[Category:20170324]]
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