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=RCA Test (Started [[Daniel:Notebook/ComboLock/2017-3-24|March 24]])= [[Daniel:Notebook/ComboLock|Back to Calendar]] ==Bgl Digestion-Excessive== <ol> <li>Bgl Digestion</li> <ol type="A"> <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="font-size:12pt;font-weight:bold" align="center" |style="background-color:#8DB4E2" width="190" height="30" | Reagent |style="background-color:#8DB4E2" width="85" | Single Rxn Vol |style="background-color:#95B3D7" width="85" | Master Mix (6.2X) |- style="font-size:12pt" | height="15" valign="bottom" | RCA Rxn | align="center" align="center" valign="bottom" | 10 | align="center" align="center" valign="bottom" | 0 |- style="background-color:#BFBFBF;font-size:12pt" | height="15" valign="bottom" | 10X Buffer 3.1 | align="center" align="center" valign="bottom" | 4 | align="center" align="center" valign="bottom" | 24.8 |- style="font-size:12pt" | height="15" valign="bottom" | Bglii | align="center" align="center" valign="bottom" | 2 | align="center" align="center" valign="bottom" | 0 |- style="background-color:#BFBFBF;font-size:12pt" | height="15" valign="bottom" | nfH2O | align="center" align="center" valign="bottom" | 24 | align="center" align="center" valign="bottom" | 148.8 |- style="font-size:12pt" | height="15" valign="bottom" | Total |style="font-weight:bold" align="center" align="center" valign="bottom" | 40 |style="font-weight:bold" align="center" align="center" valign="bottom" | 173.6 |} <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>4 hr 37C; '''When this cycle starts add 2 uL Bglii'''</li> <li>Heat kill with 65C for 20 min</li></ol> </ol> <li>TBE Gel</li> <ol type="A"> <li>Mix 60 uL 10X TBE and 20 uL 6X dye</li> <li>Aliquot 10 uL on to parafilm per sample, and 10 uL TBE + 2 uL 6X dye to ladder aliquot</li> <li>Add 4 uL reaction or 1.5 uL ladder to appropriate aliquot</li> <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:2017-04-05_-RCATest-CPLP-ssSynthesis-sssThenBgl4hr.png|500px]] [[Category:ComboLock]] [[Category:20170324]]
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