Matt:LabNotes/2014-1-27: Difference between revisions

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====Special Notes====
====Special Notes====
*Saved new images (C7, D7, D9, E9) in Jan172014 Folder
*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
*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
**The samples were left on microscope stage with computer monitor on for 2 hours
**Upon return, much more signal was seen
**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
 
{| {{table}}
| align="center" style="background:#f0f0f0;"|'''Initial Image'''
| align="center" style="background:#f0f0f0;"|'''After 2 Hours'''
|-
| [[File:C7_ch00_MIP.png | 450px]]||[[File:C7_after2hrs_ch00_MIP.png | 450px]]
|}
 
====Results====
*"After" means after hybridizing padlock probes, ampligase, and/or exonuclease I/III
**For each well see: [[Matt:LabNotes/2014-1-15#Procedure]]
{| {{table}}
| align="center" style="background:#f0f0f0;"|'''Wells'''
| align="center" style="background:#f0f0f0;"|'''HybridizePP'''
| align="center" style="background:#f0f0f0;"|'''After'''
| align="center" style="background:#f0f0f0;"|'''After Overlap HybridizePP'''
| align="center" style="background:#f0f0f0;"|'''Non-Specific'''
| align="center" style="background:#f0f0f0;"|'''After Overlap Nonspec'''
| align="center" style="background:#f0f0f0;"|'''HybridizePP Overlap Nonspec'''
|-
| C7||20,311||18,649||16,753||24,436||16,009||17,698
|-
| D7||5,524||5,296||4,648||6,597||4,660||4,790
|-
| E7||16,227||10,546||8,853||15,455||8,958||12,715
|-
| F7||13,893||11,491||9,205||14,449||9,253||10,539
|-
| D9||4,179||752||703||946||626||829
|-
| E9||11,059||8,837||6,947||12,779||6,701||7,424
|}
 
====Conclusions====
*Why would there be MORE signal for non-specific than HybridizePP?
**Nonsensical!
*Since most of non-specific signal overlaps with HybridizePP and "After" it's not necessarily non-specific
**Could it be that Shifted FISSEQ dye probe hybridizes to primary rolonies?!
***The Shifted FISSEQ 18nt (GGATATCGGGAAGCTGAA) has 6nt exactly matching RT primer (TCTCGGGAACGCTGAAGA): GCTGAA
 
 
Continue exploring this problem: [[Matt:LabNotes/2014-1-28]]

Latest revision as of 23:47, 28 January 2014

Pre-circularizing ppMALAT1 with Padlock Probe Capture[edit]

  • 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[edit]

  • 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[edit]

  • 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[edit]

  • 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[edit]

  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[edit]

  • 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

Results[edit]

Wells HybridizePP After After Overlap HybridizePP Non-Specific After Overlap Nonspec HybridizePP Overlap Nonspec
C7 20,311 18,649 16,753 24,436 16,009 17,698
D7 5,524 5,296 4,648 6,597 4,660 4,790
E7 16,227 10,546 8,853 15,455 8,958 12,715
F7 13,893 11,491 9,205 14,449 9,253 10,539
D9 4,179 752 703 946 626 829
E9 11,059 8,837 6,947 12,779 6,701 7,424

Conclusions[edit]

  • Why would there be MORE signal for non-specific than HybridizePP?
    • Nonsensical!
  • Since most of non-specific signal overlaps with HybridizePP and "After" it's not necessarily non-specific
    • Could it be that Shifted FISSEQ dye probe hybridizes to primary rolonies?!
      • The Shifted FISSEQ 18nt (GGATATCGGGAAGCTGAA) has 6nt exactly matching RT primer (TCTCGGGAACGCTGAAGA): GCTGAA


Continue exploring this problem: Matt:LabNotes/2014-1-28