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=RCA Test= [[Daniel:Notebook/ComboLock|Back to Calendar]] This experiment is to test the effectiveness of RCA so that I can use the most efficient method of amplification for the cell-based experiments. This builds on the [[Daniel:Notebook/ComboLock/2017-3-17|T4 experiments]] and the [[Daniel:Notebook/ComboLock/2017-3-22|circular product production]]. I will be using that circular product to analyze the effectiveness of RCA. ==Linear Product Production (Continued from [[Daniel:Notebook/ComboLock/2017-3-24|yesterday]])== <ol start="3"> <li>TBE gel</li> <ol type="A"> <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-20170325-LinearProduct.png|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></ol> [[Image:2017-03-25-LinearProductProduction.png|400px]] Still too much circular product present. I'll try one more time to make it linear. ==Linear Product Production== This time we'll start with the circular product and chop it. <ol start="3"> <li>Bglii digest</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="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" | Circular Product (1X) | align="center" align="center" valign="bottom" | 4 |- style="background-color:#BFBFBF;font-size:12pt" | height="15" valign="bottom" | Bglii Cutter Primer (100 uM) | align="center" align="center" valign="bottom" | 2 |- 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" | 11 |- 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>4 hr 37C; '''When this cycle starts add 1 uL Bglii'''</li> <li>Heat kill with 65C for 20 min</li></ol> </ol> Continued [[Daniel:Notebook/ComboLock/2017-3-26|tomorrow]] [[Category:ComboLock]] [[Category:20170322]]
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