Matt:LabNotes/2014-1-27

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Revision as of 18:26, 28 January 2014 by >Mzcai (→‎Special Notes)
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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

  1. Strip with 80% formamide for 10min @50C
    1. Rinse twice with H2O
  2. Image 488 channel
  3. Add 50ul 2uM Shifted FISSEQ preheated to 60C (1ul 200uM Dye + 30ul 100% formamide + 10ul 20X SSC + 59ul H2O)
    1. Incubate RT for 15min
    2. Wash 2 times with 2X SSC
  4. Image 488 channel

Special Notes

  • Saved new images (C7, D7, D9, E9) in Jan172014 Folder
  • During imaging, first imaged C7 and so only nuclei fluorescing 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 first checked D7 and saw lots of signal, so I checked C7 again and much more signal was seen
    • All wells were then checked after these 2 hours
Initial Image After 2 Hours
File:C7 ch00 MIP.png File:C7 after2hrs ch00 MIP.png