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=RCA Test (Started [[Daniel:Notebook/ComboLock/2017-3-24|March 24]])= [[Daniel:Notebook/ComboLock|Back to Calendar]] ==Second Strand Synthesis part II== Since the second strand synthesis from [[Daniel:Notebook/ComboLock/2017-4-3|Monday]] worked well, I'm going to repeat the experiment but using the NEB 3 hr and 6 hr samples (X and P, respectively). I won't bother with the Bgl digested ones as I'll just digest after. <ol> <li>qPCR-Second strand synthesis</li> <ol type="A"> <li>Make the following 14.2X master mix</li> <ol type="a"> <li>14.2 uL AmpF-6.4Sol (10 uM)</li> <li>284 uL Kapa SYBR 2X Master Mix</li> <li>241.4 uL nfH2O</li></ol> <li>Aliquot 38 uL master mix into each lane</li> <li>Add 2 uL sample according to plate layout</li> [[Image:PlateLayout-qPCR-20170405-RCATest-CPLP-ssSynthesis2.png|600px]] <li>Run the following protocol</li> <ol type="a"> <li>95C for 2 min</li> <li>95C for 5 sec</li> <li>55C for 30 sec</li> <li>72C for 45 sec</li> <li>Goto (b) x20</li> <li>72C for 2 min</li></ol> </ol> <li>Bgl Digestion</li> <ol type="A"> <li>Make the following reactions; '''Do not add Bglii yet'''</li> {| class="wikitable" <hiddentext>generated with [[:de:Wikipedia:Helferlein/VBA-Macro for EXCEL tableconversion]] V1.8</hiddentext> |- style="font-size:12pt;font-weight:bold" align="center" |style="background-color:#8DB4E2" width="180" height="30" | Reagent |style="background-color:#8DB4E2" width="85" | Single Rxn Vol |style="background-color:#95B3D7" width="85" | Master Mix (12.2X) |- style="font-size:12pt" | height="15" valign="bottom" | RCA Rxn | align="center" align="center" valign="bottom" | 8 | align="center" align="center" valign="bottom" | 0 |- style="background-color:#BFBFBF;font-size:12pt" | height="15" valign="bottom" | 10X Buffer 3.1 | align="center" align="center" valign="bottom" | 2 | align="center" align="center" valign="bottom" | 24.4 |- style="font-size:12pt" | height="15" valign="bottom" | Bglii | align="center" align="center" valign="bottom" | 1 | align="center" align="center" valign="bottom" | 0 |- style="background-color:#BFBFBF;font-size:12pt" | height="15" valign="bottom" | nfH2O | align="center" align="center" valign="bottom" | 9 | align="center" align="center" valign="bottom" | 111.6 |- style="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" | 136 |} <li>Aliquot 11 uL master mix into each sample</li> <li>Add appropriate sample and 1 uL Bglii to each</li> <li>Incubate 1 hr 37C</li> <li>Heat kill with 65C for 20 min</li> </ol> <li>TBE Gel-PreBgl</li> <ol type="A"> <li>Mix 96 uL 10X TBE and 24 uL 6X dye</li> <li>Aliquot 10 uL on to parafilm per sample</li> <li>Add 2 uL reaction or 1.5 uL ladder to appropriate aliquot</li> <li>Add 10 uL mix to gel lanes</li> [[Image:GelLanes-20170405-RCATest-CPLP-PostqPCR-PreBgl.png|400px]] <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> <li>TBE Gel-PostBgl</li> <ol type="A"> <li>Mix 120 uL 10X TBE and 40 uL 6X dye</li> <li>Aliquot 10 uL on to parafilm per sample, and 10 uL TBE + 2 uL 6X dye to ladder aliquot</li> <li>Add 4 uL reaction or 1.5 uL ladder to appropriate aliquot</li> <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> ===Results=== <gallery perrow=3 heights=300px widths=300px> File:20170405-qPCR-RCATest-CPLP-ssSynthesis2.png|qPCR curves File:2017-04-05_-RCATest-CPLP-ssSynthesis2-PreBgl.png|Gel image-pre Bgl digestion File:2017-04-05_-RCATest-CPLP-ssSynthesis2-PostBgl.png|Gel image-post Bgl digestion </gallery> [[Category:ComboLock]] [[Category:20170324]]
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