Editing
Matt:LabNotes/2016-11-16
(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!
===Day 1=== #Prepare plastic culture dish with 12x17mm hole, tweezers, large plastic dish, and 50C hot plate #*EtOH and UV sterilize #Made 4% PFA in 1X PBS #*10ml 16% PFA + 4ml 10X PBS + 26ml H2O #Take out brain section from -80C and dry on 50C hot plate for 3min #Submerge coverslip in 5ml 4% PFA in petri dish for 15min at 37C #Wash twice with cold 1X SSPE by submerging #Add 0.25% TX-100 in 2XSSPE and incubate 10min at RT #Wash with cold nf-H2O three times '''and check for degradation''' #Add 0.01% Pepsin in 0.1N HCl and incubate for 90sec at 37C #*2ul 1%Pepsin + 10ul 2N HCl + 188ul H2O #Wash with nf-1X PBS three times '''and check for degradation''' #*First try: ALL the tissue sloughed off by the end of 3rd wash #*Second try: Decreased pepsin time to 60sec and only the areas circled in red in image below sloughed off so continued #**Polymerization of gel was successful but when trying to remove coverslip from glass slide the coverslip cracked #*Third try: With same pepsin time of 60sec all tissue sloughed off #*Fourth try: Skipped pepsin step but then dropped coverslip face down on bench... SHIT! [[File:IMG_0297.jpg|350px]] #Aspirate any liquid and place a glass slide with 50um spacer over the coverslip with tissue #*Secure in plastic jig #Add 50ul gel casting mix '''filtered and degassed''' #Seal in plastic bag and vacuum out air before filling with argon #Let sit at RT for 30min #Aspirate non-polymerized gel and wash once with 1X PBS #Attach coverslip to bottom of petri dish #Aspirate non-polymerized gel and wash once with 1X PBS #Prepare SplintR Mix #*Preheat Padlock Probes then snap cool {| {{table}} | align="center" style="background:#f0f0f0;"|'''Component''' | align="center" style="background:#f0f0f0;"|'''Volume''' |- | 10X SplintR Buffer||10 |- | [[Matt:LabNotes/2016-11-10#Qubit_ssDNA|CA12kOct2016_V4 160nM]]||60 |- | H2O||30 |- | Total||100 |} *Padlock probes are mixed with SplintR Buffer but since they weren't used put in 4C box labeled "Matt" **May need to spike in ATP next time it is used <!-- #Move sample to 60C oven and slowly decreases to 55C and held for ~24hr ===Day 2=== #Wash with 1X PBS twice preheated to 55C #*Sample was dry in center, unusual amount of condensation on lid. Image borders of tissue should be okay. #Add SplintR Enzyme and incubate at 37C for 20min #*20ul 10X Buffer + 6ul SplintR + 174ul H2O #Wash with 1X PBS twice #Add 200ul (1ul 200uM FISSEQ_ppRCA + 199ul 2X SSC + 30% formamide) pre-heated to 65C and incubate 1hr at 55C #Aspirate and wash with 2X SSC, then 1X SSC, and finally 1X PBS #Prepare RCA reaction mix on ice {| {{table}} | align="center" style="background:#f0f0f0;"|'''Component''' | align="center" style="background:#f0f0f0;"|'''Volume''' |- | H2O||174 |- | 10X Phi29 Buffer||20 |- | 25mM dNTP||2 |- | 4mM aa-dUTP||2 |- | Phi 29 (low conc)||2 |- | Total||200 |} #Add RCA mix and incubate at 30C overnight (~15hrs) ===Day 3=== #Wash with 1X PBS once #Add 200ul BS(PEG)9 (4ul BS(PEG)9 + 196ul 1X PBS) and incubate at RT for 1hr #Wash with 1X PBS twice #Add 1M Tris pH 8.0 and incubate at RT for 30min #Wash with 1X PBS twice ==Imaging== #Add 0.5uM FISGA_Adpt in 30% formamide + 2X SSC preheated to 75C #Incubate for 10min at RT #Wash with 2X SSC twice ==Results== ==Conclusion==
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