Matt:LabNotes/2014-1-27
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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
Lane | Sample | 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
Testing non-specific binding of Shifted FISSEQ
- Continued from: Matt:LabNotes/2014-1-17
- On 1/17/2014 I tested the negative control wells for non-specific binding and found considerable amounts
- Today I checked the other 4 wells (C7, D7, D9, E9) following the exact same protocol
Procedure
- Strip with 80% formamide for 10min @50C
- Rinse twice with H2O
- Image 488 channel
- Add 50ul 2uM Shifted FISSEQ preheated to 60C (1ul 200uM Dye + 30ul 100% formamide + 10ul 20X SSC + 59ul H2O)
- Incubate RT for 15min
- Wash 2 times with 2X SSC
- Image 488 channel
Special Notes
- Saved new images (C7, D7, D9, E9) in Jan172014 Folder
- During imaging, first imaged C7 but then had to go work on something else for 2 hours
- The samples were left on microscope stage with computer monitor on for 2 hours
- Upon return, much more signal was seen