Matt:LabNotes/2014-1-27: Difference between revisions
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**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 first checked D7 and saw lots of signal, so I checked C7 again and 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}} | {| {{table}} | ||
Line 60: | Line 61: | ||
| [[File:C7_ch00_MIP.png | 450px]]||[[File:C7_after2hrs_ch00_MIP.png | 450px]] | | [[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]
- 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[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]
- "After" means after hybridizing padlock probes, ampligase, and/or exonuclease I/III
- For each well see: Matt:LabNotes/2014-1-15#Procedure
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
- Could it be that Shifted FISSEQ dye probe hybridizes to primary rolonies?!
Continue exploring this problem: Matt:LabNotes/2014-1-28