Editing
Daniel:Notebook/RNAFISH/2015-2-24
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!
=Linnarsson Protocol= [[Daniel:Notebook/RNAFISH|Back to Calendar]] I'll be starting work today on the Linnarsson lab protocol for doing RNA FISH on tissue sections. The protocol is given to me from Simone, one of their main RNA FISH researchers, who visited our lab in Dec 2014/Jan 2015. ==Buffer Prep== The protocol uses several solutions that are prepared beforehand and aliquoted for later use. Most of these need to only be done once. *'''E Coli tRNA 20 mg/mL''' **tRNA 100 mg **RNAse free H2O 2.5 mL **aliquot into 500 uL increments and store at -20C *'''DAPI stock solution''' **RNAse free water 500 uL **DAPI powder 10 mg **aliquot into 5 20 uL increments and store at -20C **store the remainder at -20C *'''DAPI ready to use''' **DAPI stock (20 mg/mL) 10 uL **nfH2O 990 uL **Store at 4C use within 1 month *'''70% Ethanol''' **700mL absolute EtOH **300 mL ddi water *'''Ribonucleoside Vanadyl complex (200mM)''' **Incubate the purchased solution in a water bath at 65C for 10 minutes **Aliquot in 100 uL and store up to 4 months at -20C *'''BSA 50 mg/mL''' **Came as 1 mL of 50 mg/mL, not going to split it NOTES: *I need fresh formamide and fresh paraformaldehyde 16% w/v I will order both of these *For now, I am using less fresh bottles ==Dye Coupling== *FOXP2-488 *GAD1-594 #Warm DMSO and bicarbonate labeling buffer and nuclease free water to room temperature #Add 5 uL DNA and 3 uL of sodium bicarbonate buffer #Denature samples for 5 minutes at 95C, then snap cool using ice box #Dissolve 1 vial of reactive dye in 2 uL solvent; vortex β₯10 seconds ##dye cannot be saved for later use. Use immediately! #Add 2 uL dye to sample #Incubate in the dark for 1 hour #Add 10 uL 3M NaOAc and 80 uL nfH20 to sample #[[Daniel:Protocols/CentriSep|Centri-Sep]] column purification after incubation ##Use TE Buffer #Ethanol Precipitation; incubate overnight #Continued [[Daniel:Notebook/RNAFISH/2015-2-25|tomorrow]]
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