Editing
Matt:LabNotes/2014-1-27
Jump to navigation
Jump to search
Warning:
You are not logged in. Your IP address will be publicly visible if you make any edits. If you
log in
or
create an account
, your edits will be attributed to your username, along with other benefits.
Anti-spam check. Do
not
fill this in!
===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 ===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 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 {| {{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]]
Summary:
Please note that all contributions to ZhangLabWiki may be edited, altered, or removed by other contributors. If you do not want your writing to be edited mercilessly, then do not submit it here.
You are also promising us that you wrote this yourself, or copied it from a public domain or similar free resource (see
ZhangLabWiki:Copyrights
for details).
Do not submit copyrighted work without permission!
Cancel
Editing help
(opens in new window)
Template used on this page:
Template:Table
(
edit
)
Navigation menu
Personal tools
Not logged in
Talk
Contributions
Create account
Log in
Namespaces
Page
Discussion
English
Views
Read
Edit
View history
More
Search
Navigation
Main Page
Current events
Recent changes
Random page
Investigators
Matt Cai
Song Chen
Eric Chu
Dinh Diep
Elizabeth Duong
Shicheng Guo
Alan Fung
Daniel Jacobsen
Blue Lake
Huy Lam
Alice Li
Andrew Richards
Brandon Sos
Chris Wei
Yan Wu
Kun Zhang
Tools
What links here
Related changes
Special pages
Page information