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==Protocol== <ol> <li>Circularization</li> <ol type ="A"> <li>Prepare (N+1)X uL Phusion mix (N samples)</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="140" height="43" | Reagent | width="85" | Stock Conc | width="85" | Final Amount | width="120" | 1x Vol (uL) |- style="font-size:12pt" | height="15" valign="bottom" | NAD+ | align="center" | 5 mM | align="center" | 40 nmol | align="center" align="center" | 8 |- style="background-color:#BFBFBF;font-size:12pt" | height="15" valign="bottom" | dNTP | align="center" | 1 mM | align="center" | 600 pmol | align="center" align="center" | 0.6 |- style="font-size:12pt" | height="15" valign="bottom" | Betaine | align="center" | 5 M | align="center" | 15 umol | align="center" align="center" | 3 |- style="background-color:#BFBFBF;font-size:12pt" | height="15" valign="bottom" | 10X AmpLigase Buffer | align="center" | 10X | align="center" | 1X | align="center" align="center" | 2 |- style="font-size:12pt" | height="15" valign="bottom" | Amp Ligase | align="center" | 5 U/uL | align="center" | 10 U | align="center" align="center" | 2 |- style="background-color:#BFBFBF;font-size:12pt" | height="30" valign="bottom" | Phusion HF DNA Polymerase | align="center" | 2000 U/mL | align="center" | 6.4U | align="center" align="center" | 3.2 |- style="font-size:12pt" | height="15" valign="bottom" | nf H2O | align="center" align="center" | | align="center" align="center" | | align="center" align="center" | 1.2 |- style="background-color:#BFBFBF;font-size:12pt" |style="font-weight:bold" height="15" valign="bottom" | Total | align="center" align="center" | | align="center" align="center" | | align="center" align="center" | 20 |} <li>Add 20 uL Phusion mix to 20 uL of reaction suspended in 1X buffer</li> <li>Incubate at 60C for 2 hours</li> <li>Heat kill the reaction by incubating for 2 minutes at 94C</li></ol> <li>Exonuclease Digestion</li> <ol type="A"> <li>Combine equal molar amounts of Exonuclease I (20U/uL) and Exonuclease III (100U/uL)</li> <li>Add 2 uL of mix to each reaction</li> <li>Incubate for 30 minutes to 2 hours at 37C</li> <li>Heat kill by incubating for 5 minutes at 94C</li></ol> <li>Rolling Circle Amplification</li> <ol type="A"> <li>Prepare (N+1)X 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:#C4D79B;font-size:12pt;font-weight:bold" align="center" | width="150" height="45" | Reagent | width="90" | Stock Concentration | width="90" | Final Conc/Amt | width="90" | uL Added in 20 uL Rxn |- style="font-size:12pt" | height="15" valign="bottom" | Template | align="center" valign="bottom" | NA | align="center" valign="bottom" | NA | align="center" align="center" valign="bottom" | 5 |- style="background-color:#BFBFBF;font-size:12pt" | height="15" valign="bottom" | RCA Primer | align="center" valign="bottom" | 10 uM | align="center" valign="bottom" | 2 uM | align="center" align="center" valign="bottom" | 4 |- style="font-size:12pt" | height="15" valign="bottom" | dNTP | align="center" valign="bottom" | 1 mM | align="center" valign="bottom" | 50 uM | align="center" align="center" valign="bottom" | 1 |- style="background-color:#BFBFBF;font-size:12pt" | height="15" valign="bottom" | 10X Buffer | align="center" valign="bottom" | 10X | align="center" valign="bottom" | 1X | align="center" align="center" valign="bottom" | 2 |- style="font-size:12pt" | height="15" valign="bottom" | Phi29 (NEB) | align="center" valign="bottom" | 10 U/uL | align="center" valign="bottom" | 10 U | align="center" align="center" valign="bottom" | 1 |- style="background-color:#BFBFBF;font-size:12pt" | height="15" valign="bottom" | nfH2O | align="center" valign="bottom" | NA | align="center" valign="bottom" | NA | align="center" align="center" valign="bottom" | 7 |- style="font-size:12pt" | height="15" valign="bottom" | Total | align="center" align="center" valign="bottom" | | align="center" align="center" valign="bottom" | |style="font-weight:bold" align="center" align="center" valign="bottom" | 20 |} <li>Add 15 uL master mix to each tube</li> <li>Add 5 uL sample to correct reaction tubes</li> <li>Incubate at 37C for 3 hours</li> <li>Heat kill by incubating at 65C for 10 minutes</li></ol> </ol>
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