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==Protocol== <ol> <li>Dephosphorylation (Bolt Oligo)</li> <ol type="A"> <li>Mix the following reaction</li> <ol type="a"> <li>4 uL Circ Latch oligo (100 uM)</li> <li>2 uL CutSmart Buffer (10X)</li> <li>2 uL rSAP enzyme</li> <li>12 uL nfH2O</li></ol> <li>Incubate at 37C for 30 minutes</li> <li>Incubate at 65 for 5 minutes</li></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" | Dephosphorylated Circ Latch (Step 1) | align="center" | 20 uM | align="center" | 100 pmol | align="center" align="center" | 5 |- style="font-size:12pt" | height="15" valign="bottom" | Padlock 0601 oligo | align="center" | 100 uM | align="center" | 100 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" | 100 pmol | align="center" align="center" | 1 |- style="font-size:12pt" | height="15" valign="bottom" | C2 positive Control oligo | align="center" | 100 uM | align="center" | 100 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" | 6 |- 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:G|400px]] <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:2017-03-24-LinearProductProduction.png|500px]] [[Category:ComboLock]] [[Category:20170322]]
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