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=Padlock0601 Test (Started [[Daniel:Notebook/ComboLock/2017-3-1|Wednesday]])= [[Daniel:Notebook/ComboLock|Back to Calendar]] ==Protocol-Part 5== <ol start="11"> <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> <li>qPCR</li> <ol type="A"> <li>Make 9.2X qPCR master mix according to following recipe</li> <ol type="a"> <li>193.2 uL nfH2O</li> <li>230 uL SYBR Fast</li> <li>9.2 uL 10 uM AmpF</li> <li>9.2 uL 10 uM AmpR-Ind20</li> </ol> <li>Add 48 uL master mix to each well</li> <li>Add 2 uL sample according to plate layout</li> [[Image:PlateLayout-20170303-Padlock0601-Bglii.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>TBE Gel</li> <ol type="A"> <li>Mix 96 uL TBE, 24 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=3 heights=300px widths=300px caption="qPCR curves and gel images for samples. See individual gel captions for more info. Note that for all gel images Post-RCA the left sample was RC amplified with normal primer and the right sample with phosphothiorate primer"> File:20170302-qPCR-Padlock0601Test.png|qPCR curves for run on 3-2-2017 File:2017-03-03-Padlock0601-PreRCA-Unpurifed.png|Gel image-PreRCA Unpurified File:2017-03-03-Padlock0601-PreRCA-Purifed.png|Gel image-PreRCA Purifed File:2017-03-03-Padlock0601-PostRCA-Unpurifed.png|Gel image-PostRCA Unpurified File:2017-03-03-Padlock0601-PostRCA-Purifed.png|Gel image-PostRCA Purified File:20170303-qPCR-Padlock0601-Bglii.png|qPCR curves for post-Bglii digestion File:2017-03-03-Padlock0601-Bglii.png|Post BglII digestion </gallery> [[Category:ComboLock]] [[Category:20170301]]
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