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=Padlock0601 Test (Started [[Daniel:Notebook/ComboLock/2017-3-1|Yesterday]])= [[Daniel:Notebook/ComboLock|Back to Calendar]] ==Protocol-Part 3== <ol start="6"> <li>Exonuclease Digestion</li> <ol type="A"> <li>Mix 15 uL Exo I (20U/uL) and 3 uL ExoIII (100U/uL)</li> <li>Add 2 uL to each sample, mix by swirling pipette tip</li> <li>Incubate for 1.5 hours at 37C</li> <li>Heat kill by incubating for 5 min at 95C</li> </ol> <li>Column Purification-Qiaquick</li> <ol type="A"> <li>Add 150 uL (5X) PB (binding buffer) to 30 uL 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 30 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>Samples will be aliquoted under following rules</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="200" height="30" | Samples | width="220" | Condition |- style="font-size:12pt" | height="15" valign="bottom" | Sample X (1-4, AB) | align="center" valign="bottom" | Unpurified |- style="background-color:#BFBFBF;font-size:12pt" | height="15" valign="bottom" | Sample Y (1-4, AB) | align="center" valign="bottom" | Purified |- style="font-size:12pt" | height="15" valign="bottom" | A Samples (1-4) | align="center" valign="bottom" | Normal Primer |- style="background-color:#BFBFBF;font-size:12pt" | height="15" valign="bottom" | B Samples (1-4) | align="center" valign="bottom" | Phosphorothiorate Primer |} <li>Prepare 8.2X 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:#8DB4E2;font-size:12pt;font-weight:bold" align="center" | width="160" height="30" | Reagent | width="85" | Single Rxn Vol | width="85" | 16.2X MM uL |- style="font-size:12pt" | height="15" valign="bottom" | Template | align="right" align="center" valign="bottom" | 4 | 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" | 0 |- style="font-size:12pt" | height="15" valign="bottom" | dNTP (1 mM) | align="right" align="center" valign="bottom" | 5 | align="right" align="center" valign="bottom" | 81 |- 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" | 32.4 |- style="font-size:12pt" | height="15" valign="bottom" | Phi29 | align="right" align="center" valign="bottom" | 1 | 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" | 6.48 |- style="font-size:12pt" | height="15" valign="bottom" | nfH2O | align="right" align="center" valign="bottom" | 5.1 | align="right" align="center" valign="bottom" | 82.62 |- style="background-color:#BFBFBF;font-size:12pt" | height="15" valign="bottom" | Total |style="font-weight:bold" align="right" align="center" valign="bottom" | 20 |style="font-weight:bold" align="right" align="center" valign="bottom" | 202.5 |} <li>Add 12.5 uL master mix to each tube</li> <li>Add 4 uL template according to table</li> <li>Add 2.5 uL correct primer according to table</li> <li>Incubate at 95 C for 5 minutes</li> <li>Lower to 55C for 15 minutes</li> <li>Lower to 30C for at least 2 minutes</li> <li>Add 1 uL Phi29 polymerase to each sample</li> <li>Incubate 3 hours at 30C</li> <li>Heat kill enzyme by incubating 10 minutes at 65C; Hold at 12C</li> </ol></ol> ==Protocol-Part 4== <ol start="9"> <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="200" height="30" | Reagent | width="85" | Single Rxn Vol | width="85" | 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" | 2 | align="center" align="center" valign="bottom" | 16.4 |- 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" | 0 |- style="font-size:12pt" | height="15" valign="bottom" | nfH2O | align="center" align="center" valign="bottom" | 10 | align="center" align="center" valign="bottom" | 82 |- 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 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 plate layout</li> [[Image:PlateLayout-20170302-Padlock0601Test.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></ol> qPCR for post-Bglii digestion and gel images for all data continued [[Daniel:Notebook/ComboLock/2017-3-3|tomorrow]] [[Category:ComboLock]] [[Category:20170301]]
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