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=Padlock0601 Test (Started [[Daniel:Notebook/ComboLock/2017-3-1|Wed March 1]])= [[Daniel:Notebook/ComboLock|Back to Calendar]] ==Protocol-Part 2 (Protocol Started [[Daniel:Notebook/ComboLock/2017-3-6|Yesterday]])== <ol start="2"> <li>BglII Digestion</li> <ol type="A"> <li>Use samples 1 (Experiment) and 4 (No phosphate) for Bglii digest</li> <li>Make the following 4.2X master mix</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="220" height="30" | Reagent | width="85" | Single Rxn Vol | width="90" | 8.2X MM uL |- style="font-size:12pt" | height="15" valign="bottom" | RCA Reaction Product | align="center" align="center" valign="bottom" | 5 | align="center" align="center" valign="bottom" | 0 |- style="background-color:#BFBFBF;font-size:12pt" | height="15" valign="bottom" | Bglii Cutter Primer (10 uM) | align="center" align="center" valign="bottom" | 0 | align="center" align="center" valign="bottom" | 0 |- style="font-size:12pt" | height="15" valign="bottom" | 10X Buffer 3.1 | align="center" align="center" valign="bottom" | 2 | align="center" align="center" valign="bottom" | 16.4 |- style="background-color:#BFBFBF;font-size:12pt" | height="15" valign="bottom" | Bglii | align="center" align="center" valign="bottom" | 1 | align="center" align="center" valign="bottom" | 8.2 |- style="font-size:12pt" | height="15" valign="bottom" | nfH2O | align="center" align="center" valign="bottom" | 12 | align="center" align="center" valign="bottom" | 98.4 |- 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" | 114.8 |} <li>Aliquot 14 uL master mix into new tubes</li> <li>Add 5 uL appropriate sample to each tube</li> <li>Use the following thermocycler program</li> <ol type="a"> <li>5 min 95C</li> <li>10 min 50C</li> <li>1 hr 37C; '''When this cycle starts add 1 uL Bglii'''</li> <li>Heat kill with 65C for 20 min</li></ol> </ol> <li>qPCR</li> <ol type="A"> <li>Make 16.2X qPCR master mix according to following recipe</li> <ol type="a"> <li>340.2 uL nfH2O</li> <li>405 uL SYBR Fast</li> <li>16.2 uL 10 uM AmpF</li> <li>16.2 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-20170307-Padlock0601-MaxRCA.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 160 uL TBE, 40 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 2 uL SYBR Gold for 3 minutes</li> <li>Rinse gel and image in gel doc</li> </ol></ol> ==Protocol-Part 3-Post Bglii== <ol start="5"> <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-Ind21</li> </ol> <li>Add 48 uL master mix to each well</li> <li>Add 2 uL sample according to plate layout</li> [[Image:PlateLayout-20170307-MaxRCA-BgliiDigest.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 2 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> File:20170307-qPCR-MaxRCA.png|qPCR curves for MaxRCA samples File:20170307-qPCR-MaxRCA-Adj.png|qPCR curves for MaxRCA samples; adjusted by constant offset to (generally) be non-negative File:2017-03-07-Padlock0601-MaxRCA-Unpurified.png|Gel image-Unpurified RCA input File:2017-03-07-Padlock0601-MaxRCA-Purified.png|Gel image-Purified RCA input File:20170307-qPCR-MaxRCA-Bglii.png|qPCR curves for MaxRCA samples post-Bglii digest File:2017-03-07-Padlock0601-MaxRCA-Bglii.png|Gel image-Post Bglii Digest </gallery> [[Category:ComboLock]] [[Category:20170301]]
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