Editing
Matt:LabNotes/2014-4-9
(section)
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!
==Efficiency of Ampligase in situ Test== *Samples are wells F3-F10 from [[Matt:LabNotes/2014-3-31]] **[[Matt:LabNotes/2014-3-31#Results | # of Rolonies at each position]] *General idea: Will use Exo I & Exo III OR Exo III only to digest non-ligased padlock probes, then use dcProbe2RevComp to hybridize to padlock probe and detect **Exo III only because it's possible Exo I will degrade all rolonies, leaving the padlock probe (even after ligation) nothing to attach to so it'll be washed away and not detected *Ligation with Ampligase should protect the padlock probes from digestion, hence comparing wells +Exo with those -Exo will show what percentage of padlock probes are ligated ===Hybridize 2uM ppMALAT1=== *Add 50ul 2uM ppMALAT1 for 21.5hr @45C (7:00pm-4:30pm ) in 1X Ampligase buffer +/- 0.5U/ul Ampligase F3-F6: -Ampligase {| {{table}} | align="center" style="background:#f0f0f0;"|'''Component''' | align="center" style="background:#f0f0f0;"|'''Volume''' |- | 10X Ampligase Buffer||20 |- | 200uM ppMALAT1||2 |- | H2O||178 |- | Total||200 |} F7-F10: + Ampligase {| {{table}} | align="center" style="background:#f0f0f0;"|'''Component''' | align="center" style="background:#f0f0f0;"|'''Volume''' |- | 10X Ampligase Buffer||20 |- | 200uM ppMALAT1||2 |- | 5 U/ul Ampligase||20 |- | H2O||158 |- | Total||200 |} ===Digestion=== *Wash with 2X SSC once *Add 50ul Exonuclease mix *Incubate @37C for 2 hrs ====Exo I/III: F4 & F8==== {| {{table}} | align="center" style="background:#f0f0f0;"|'''Component''' | align="center" style="background:#f0f0f0;"|'''Volume''' |- | 10X Exo III Buffer||10 |- | Exo I||5 |- | Exo III||5 |- | H2O||80 |- | Total||100 |} ====Exo III: F3 & F9==== {| {{table}} | align="center" style="background:#f0f0f0;"|'''Component''' | align="center" style="background:#f0f0f0;"|'''Volume''' |- | 10X Exo III Buffer||10 |- | Exo III||5 |- | H2O||85 |- | Total||100 |} ====Control: F5, F6, F7 & F10==== {| {{table}} | align="center" style="background:#f0f0f0;"|'''Component''' | align="center" style="background:#f0f0f0;"|'''Volume''' |- | 10X Exo III Buffer||20 |- | H2O||180 |- | Total||200 |} ===Detect=== *Wash with 2X SSC twice *Add 50ul 1uM dcProbe2RevComp-Cy3 (ATTO 488 dye) **Pre-heated to 75C then snap-cooled before adding *Wash with 2X SSC twice *Images saved in '''4-10-2014''' ===Results=== {| {{table}} | align="center" style="background:#f0f0f0;"|'''Position''' | align="center" style="background:#f0f0f0;"|'''Well''' | align="center" style="background:#f0f0f0;"|'''Ampligase''' | align="center" style="background:#f0f0f0;"|'''Digestion''' | align="center" style="background:#f0f0f0;"|'''Hybridized Padlock Probes''' | align="center" style="background:#f0f0f0;"|'''Rolonies''' | align="center" style="background:#f0f0f0;"|'''%''' |- | 5||F3||-||Exo III||203||15,805||1.28 |- | 6||F4||-||Exo I/III||146||7,253||2.01 |- | 7||F5||-||-||302||7,754||3.89 |- | 8||F6||-||-||230||6,210||3.70 |- | 9||F7||+||-||71||14,548||0.49 |- | 10||F8||+||Exo I/III||0||13,323||0.00 |- | 11||F9||+||Exo III||174||27,904||0.62 |- | 12||F10||+||-||195||22,660||0.86 |} *Using bkg_mult = 3 because a lot of features weren't detected as "polonies" *Also summed the intensity values of each pixel in an image since it should correlate more with number of tagged padlock probes rather than # of rolonies tagged by padlock probes {| {{table}} | align="center" style="background:#f0f0f0;"|'''Position''' | align="center" style="background:#f0f0f0;"|'''Well''' | align="center" style="background:#f0f0f0;"|'''Ampligase''' | align="center" style="background:#f0f0f0;"|'''Digestion''' | align="center" style="background:#f0f0f0;"|'''Hybridized Padlock Probes''' | align="center" style="background:#f0f0f0;"|'''Rolonies''' | align="center" style="background:#f0f0f0;"|'''%''' | align="center" style="background:#f0f0f0;"|'''Intensity*Area''' |- | 5||F3||-||Exo III||251||15,805||1.59|| 34,553,526 |- | 6||F4||-||Exo I/III||191||7,253||2.63|| 24,825,840 |- | 7||F5||-||-||311||7,754||4.01|| 53,719,236 |- | 8||F6||-||-||310||6,210||4.99|| 47,879,730 |- | 9||F7||+||-||107||14,548||0.74|| 31,531,795 |- | 10||F8||+||Exo I/III||0||13,323||0.00|| 19,118,820 |- | 11||F9||+||Exo III||222||27,904||0.80|| 42,094,484 |- | 12||F10||+||-||245||22,660||1.08|| 49,782,492 |} [[File:20140410_EfficiencyAmpligaseinsituTest.JPG]] ===Conclusion=== *Exonuclease digestion did not seem complete **Possibly adding 2uM of ppMALAT1 is too much for Exo I/III to handle in 2hrs? but doubt it **Use [[Matt:LabNotes/2014-4-6 | 10nM ppMALAT1]] next time to match the Exo I/III test *Because -Ampligase wells with +Exo still detected signal, cannot draw any conclusions about +Ampligase wells *The average % of detected rolonies of +Ampligase wells is less than average of -Ampligase wells: **Does Ampligase decrease the efficiency % of hybridization? **Or is it because the +Ampligase wells had more rolonies to start with? ***If that were true, it would imply ppMALAT1 is limiting reagent, which is unlikely given 2uM **Try adding 50% glycerol in -Ampligase wells next time to better mimic +Ampligase wells
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)
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