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Daniel:Notebook/ComboLock/2017-3-22
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=Circular Product Production= [[Daniel:Notebook/ComboLock|Back to Calendar]] The goal of this will be to produce a large amount of circular product for use in testing. ==Protocol== <ol> <li>Ligation</li> <ol type="A"> <li>Make reaction mixture according to table; '''Do not add ligase yet'''</li> {| class="wikitable" <hiddentext>generated with [[:de:Wikipedia:Helferlein/VBA-Macro for EXCEL tableconversion]] V1.8</hiddentext> |- style="background-color:#66FFFF;font-size:12pt;font-weight:bold" align="center" | width="220" height="36" | Reagent | width="100" | Stock Conc | width="100" | Final Conc./Amount | width="106" | uL added |- style="font-size:12pt" | height="15" valign="bottom" | T4 Ligase Buffer | align="center" | 5X | align="center" | 1X | align="center" align="center" | 4 |- style="background-color:#BFBFBF;font-size:12pt" | height="15" valign="bottom" | PosCon Circ Latch Oligo | align="center" | 100 uM | align="center" | 200 pmol | align="center" align="center" | 1 |- style="font-size:12pt" | height="15" valign="bottom" | Padlock 0601 oligo | align="center" | 100 uM | align="center" | 200 pmol | align="center" align="center" | 1 |- style="background-color:#BFBFBF;font-size:12pt" | height="15" valign="bottom" | C4 Positive Control oligo | align="center" | 100 uM | align="center" | 200 pmol | align="center" align="center" | 1 |- style="font-size:12pt" | height="15" valign="bottom" | C2 positive Control oligo | align="center" | 100 uM | align="center" | 200 pmol | align="center" align="center" | 1 |- style="background-color:#BFBFBF;font-size:12pt" | height="15" valign="bottom" | T4 DNA Ligase | align="center" | 5 U/uL | align="center" | 5 U | align="center" align="center" | 1 |- style="font-size:12pt" | height="15" valign="bottom" | nfH2O | align="center" | NA | align="center" | NA | align="center" align="center" | 11 |- style="background-color:#BFBFBF;font-size:12pt" |style="font-weight:bold" height="15" valign="bottom" | Total | align="center" align="center" | | align="center" align="center" | |style="font-weight:bold" align="center" align="center" | 20 |} <li>Use following thermocycler program</li> <ol type="a"> <li>Heat mixture to 95C for 5 min</li> <li>Lower temp by 0.2C per second to 55C</li> <li>Hold mixture at 55C for 15 minutes</li> <li>Lower temp to 20C by reducing 0.2C per second</li> <li>Hold at 20C</li></ol> <li>Add 1 uL T4 DNA ligase</li> <li>Incubate at 20C for 30 minutes</li> <li>Heat kill enzyme by incubating at 65C for 15 minutes</li> <li>Add 20 uL nfH2O</li></ol> <li>TBE gel</li> <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-20170322-Circ.png|400px|Gel lanes]] <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> [[Image:2017-03-22-CPProduction.png|500px]] ==Protocol Part 2== That was unexpected. From what I'm seeing on [http://www.bioinformatics.nl/molbi/SimpleCloningLab/electrophoresis.htm the internet], this might be a thing. Circular DNA looks to appear much higher on the gel than it should. I can try and Bglii digest to be sure. <ol start="3"> <li>Bglii digest</li> <ol type="A"> <li>Use samples 1 (Experiment) and 4 (No phosphate) for Bglii digest</li> <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="background-color:#8DB4E2;font-size:12pt;font-weight:bold" align="center" | width="220" height="30" | Reagent | width="105" | Single Rxn Vol |- style="font-size:12pt" | height="15" valign="bottom" | Ligation Reaction Product | align="center" align="center" valign="bottom" | 4 |- style="background-color:#BFBFBF;font-size:12pt" | height="15" valign="bottom" | Bglii Cutter Primer (10 uM) | align="center" align="center" valign="bottom" | 5 |- style="font-size:12pt" | height="15" valign="bottom" | 10X Buffer 3.1 | align="center" align="center" valign="bottom" | 2 |- style="background-color:#BFBFBF;font-size:12pt" | height="15" valign="bottom" | Bglii | align="center" align="center" valign="bottom" | 1 |- style="font-size:12pt" | height="15" valign="bottom" | nfH2O | align="center" align="center" valign="bottom" | 8 |- style="background-color:#BFBFBF;font-size:12pt" | height="15" valign="bottom" | Total |style="font-weight:bold" align="center" align="center" valign="bottom" | 20 |} <li>Use the following thermocycler program</li> <ol type="a"> <li>5 min 95C</li> <li>Ramp to 50C at 0.2C/s</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>Qiaquick Column Purification (Circular product reaction from part 1)</li> <ol type="A"> <li>Add 200 uL PB to sample</li> <li>Centrifuge at 13000rpm for 1 minute</li> <li>Dump flow through and add 700 uL PE buffer</li> <li>Centrifuge at 13000rpm for 1 minute</li> <li>Dump flow through and add 700 uL PE buffer</li> <li>Centrifuge at 13000rpm for 1 minute</li> <li>Dump flow through and centrifuge empty for 2 min at 13000 rpm</li> <li>Add 50 uL nfH2O and centrifuge for 2 min at 13000 rpm</li></ol> <li>TBE Gel II</li> <ol type="A"> <li>Mix 80 uL TBE and 20 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:GelLanes2-20170322-Circ.png|400px|Gel lanes]] <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:2017-03-22-CPProduction-2.png|500px]] [[Category:ComboLock]] [[Category:20170322]]
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