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===Pre-circularizing ppMALAT1 with Padlock Probe Capture=== *I want to create circularized ppMALAT1 to use as a positive control for in situ ampligase reaction *Previous efforts using CircLigaseII have failed for unknown reasons *Since CircLigase doesn't work, Alan did a padlock probe capture in vitro with ampligase and target template ordered from IDT (45nt): [[AlanFung:LabNotes/Capturing/2014-1-23]] ====TBU Gel==== *I ran a denaturing gel to verify the circularization of ppMALAT1 *Gel sample: sample '''TBU Gel''' {| {{table}} | align="center" style="background:#f0f0f0;"|'''Lane''' | align="center" style="background:#f0f0f0;"|'''Sample''' | align="center" style="background:#f0f0f0;"|'''Volume (ul)''' |- | 1||Low Mass Ladder||0.5 |- | 3||100uM 90nt linear ssDNA||0.5 |- | 4||ppMALAT1 capture product||2 |- | 5||ppMALAT1 capture product PCR'd||2 |} [[File:2014-01-27_Pre-Circularize_ampligase_ppMALAT1.jpg]] *Lane 3 was overloaded with DNA causing the smear *Lane 4 with the capture product has a faint band at ~100nt range **ppMALAT1 is 92nt but circularized DNA known to run a bit slower if large and faster if small (ie. <50nt) *Lane 5 with the amplicon has many bands characteristic of PCR of circular template ====Conclusion==== *Ampligase + template can successfully circularize ppMALAT1 **but the efficiency may not be optimal *Use Qubit to quantify amount of circularized ppMALAT1 and calculate capture efficiency *Possibly will have to run many more capture reactions to create enough positive controls
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