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===PCR protocols (Note, bolded steps are PCR repeat cycles)=== <ol> <li>Church Lab Protocol</li> <ol type="A"> <li>96 uL 10X PCR buffer</li> <li>360 uL 50 mM MgCl2</li> <li>135 uL 25 mM (each) dNTP</li> <li>6.0 uL 2 mM PR1R-S</li> <li>60 uL MyOne beads in TE, pre-loaded with former primer</li> <li>54 uL 5 U/uL hot-start Taq</li> <li>1.0 uL template DNA (??? concentration)</li> <li>248 uL nfH2O</li> <li>10 min 94C; '''15 sec 94 C'''; '''30 sec 57C'''; '''75 sec 70C'''; '''goto x119'''; 2 min 70 C; 4C forever</li> </ol> <li>Shao et al Protocol</li> <ol type="A"> <li>0.4 umol/L each primer</li> <li>3.5 mmol/L MgCl2</li> <li>0.4 mmol/L each dNTP</li> <li>0.125 unit/L Taq polymerase</li> <li>0.01 pmol/mL ssDNA template</li> <li>nfH2O to 100 uL</li> <li>2 min 94C; '''30 sec 94 C'''; '''30 sec 65C'''; '''30 sec 72C'''</li> </ol> <li>Murgha et al</li> <ol type="A"> <li>2.5 fmol template</li> <li>0.5 uM each primer</li> <li>0.2 mM each dNTP</li> <li>0.5 ug/uL bovine serum albumin</li> <li>4 Units of Phusion HS polymerase and 1x GC rich buffer</li> <li>nfH2O to 100 uL</li> <li>2 min 98C; '''15 sec 98 C'''; '''25 sec (Tm-2) C'''; '''25 sec 72C'''; '''goto x30'''; 5 min 72 C</li> </ol> <li>Ion Torrent Protocol</li> <ol type="A"> <li>Use the following components</li> {| class="wikitable" <hiddentext>generated with [[:de:Wikipedia:Helferlein/VBA-Macro for EXCEL tableconversion]] V1.8</hiddentext> |- style="background-color:#6666FF;font-size:12pt;font-weight:bold" align="center" | width="200" height="30" | Component | width="100" | Volume | width="110" | Final Concentration |- style="font-size:12pt" | height="15" valign="bottom" | 10X PCR Buffer | align="center" align="center" valign="bottom" | 15 | align="center" valign="bottom" | 1X |- style="background-color:#BFBFBF;font-size:12pt" | height="15" valign="bottom" | 25mM dNTPs | align="center" align="center" valign="bottom" | 15 | align="center" valign="bottom" | 2.5 mM |- style="font-size:12pt" | height="15" valign="bottom" | 1 mM MgCl2 | align="center" align="center" valign="bottom" | 0.5 | align="center" valign="bottom" | 3.5 mM |- style="background-color:#BFBFBF;font-size:12pt" | height="15" valign="bottom" | 100 uM PrimerA | align="center" align="center" valign="bottom" | 13.5 | align="center" valign="bottom" | 9 uM |- style="font-size:12pt" | height="15" valign="bottom" | 10 uM Primer B | align="center" align="center" valign="bottom" | 1.5 | align="center" valign="bottom" | 0.1 uM |- style="background-color:#BFBFBF;font-size:12pt" | height="15" valign="bottom" | SNAPPS (hydrogel beads, 2M/uL) | align="center" align="center" valign="bottom" | 10 | align="center" valign="bottom" | 50 Million |- style="font-size:12pt" | height="15" valign="bottom" | CAC Polymerase (5 U/uL) | align="center" align="center" valign="bottom" | 18 | align="center" valign="bottom" | 90 Units |- style="background-color:#BFBFBF;font-size:12pt" | height="15" valign="bottom" | DNA template 2nd Dilution | align="center" align="center" valign="bottom" | 10 | align="center" valign="bottom" | 0.12 pM |- style="font-size:12pt" | height="15" valign="bottom" | 1 M Ammonium Sulfate | align="center" align="center" valign="bottom" | 0.75 | align="center" valign="bottom" | 5 mM |- style="background-color:#BFBFBF;font-size:12pt" | height="15" valign="bottom" | 1 mM Spermidine | align="center" align="center" valign="bottom" | 15 | align="center" valign="bottom" | 100 uM |- style="font-size:12pt" | height="15" valign="bottom" | TIPP | align="center" align="center" valign="bottom" | 0.3 | align="center" valign="bottom" | NA |- style="background-color:#BFBFBF;font-size:12pt" | height="15" valign="bottom" | Water | align="center" align="center" valign="bottom" | 64.7 | align="center" valign="bottom" | NA |- style="font-size:12pt" | height="15" valign="bottom" | Total Volume |style="font-weight:bold" align="center" align="center" valign="bottom" | 150 | align="center" align="center" valign="bottom" | |} </ol> </ol>
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