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=Overnight Capture= So based on the results above, I'd say that the latch and padlock are not both finding their targets at the same time, which is leading to a situation where nothing works. So I'm going to repeat the experiment but with an overnight capture step that will give everything time to settle. I'll incubate at 60C overnight for capture (The Tm of the adapters is designed to 65 btw). Tomorrow I will add the phusion reaction (Phusion polymerase + Amp Ligase) for 2 hours without taking the samples off the incubator. ==Sample Matrix== {| class="wikitable" <hiddentext>generated with [[:de:Wikipedia:Helferlein/VBA-Macro for EXCEL tableconversion]] V1.8</hiddentext> |- style="background-color:#CCC0DA;font-size:12pt;font-weight:bold" align="center" | width="150" height="34" | Sample | width="120" | Condition |- style="font-size:12pt" | height="15" valign="bottom" | Sample 1 (AB) | align="center" valign="bottom" | Normal |- style="background-color:#BFBFBF;font-size:12pt" | height="15" valign="bottom" | Sample 2 (AB) | align="center" valign="bottom" | No Template |- style="font-size:12pt" | height="15" valign="bottom" | Sample 3 (AB) | align="center" valign="bottom" | No C probes |} ==Protocol== <ol> <li>Template-Bead Binding</li> <ol type="A"> <li>Suspend 2 uL (8 ug) beads per sample in 100 uL wash buffer </li> <li>Apply magnet for 30 sec and remove supernatant</li> <li>Add 2 uL 10 uM template oligo per sample to bead solution; incubate at RT for 5 min</li> <li>Wash beads with 100 uL wash buffer; vortex to suspend; apply magnet and remove supernatant</li> </ol> <li>C Probe Hybridization</li> <ol type="A"> <li>Combine 1 uL of each C probe (10 uM stock) and 3 uL bead wash buffer into a 0.2 mL tube</li> <li>Heat C probes to 90C for 5 min; Chill probes on ice to quench</li> <li>Add 5 uL probe mixture to beads</li> <li>Incubate at 40C for 1 hour with agitation</li> <li>Wash beads with 100 uL wash buffer; apply magnet and remove supernatant</li> <li>Repeat wash step above</li> <li>Add 100 uL cold Low Salt Buffer; apply magnet and remove supernatant</li> </ol> <li>Latch and Padlock Hybridization</li> <ol type="A"> <li>Add following to a fresh 0.2 mL tube</li> <ol type="a"> <li>12.2 uL Latch0001, 10 uM stock</li> <li>12.2 uL Padlock0201, 10 uM stock</li> <li>12.2 uL Amp10X buffer</li> <li>85.4 uL nfH2O</li></ol> <li>Incubate at 60C overnight</li></ol> Continued [[Daniel:Notebook/ComboLock/2016-12-16|tomorrow]] [[Category:ComboLock]] [[Category:20161213]] [[Category:20161215]]
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