Editing
Matt:LabNotes/Polyacrylamide Gel Protocol
(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!
=Polyacrylamide Gel Protocol= *Based on [[Daniel:Protocols/SlidePAGE|Dan's protocol he learned from Dr. Zhang's Harvard protocol]] ==Purpose and General Procedure== *Make gel to cover and mechanically stabilize brain tissue section for DARTFISH to prevent tissue degradation *Instead of using dessicated bind-silane treated slides we have to use vectabond-treated cover glass in a tissue culture dish with a hydrated tissue section **Due to thickness of culture dish (~1mm) no way to use coverslip to prevent exposure to air while making <1mm thick gel ***Can try using mineral oil to cover instead *Not using DATD (cross-linker) in Acrylamide/Bis mix ==Materials== *10% BSA ([http://www.sigmaaldrich.com/catalog/product/sigma/a1595?lang=en®ion=US Sigma Alrich Website] *40% Acrylamide *40% Acrylamide/Bis (19:1) *Syringe and 0.22 micron filter *Ammonium persulfate (APS) *TEMED *Dessicator ==Protocol== #Turn on UV lamp in AirClean hood for 15 minutes. #Prepare fresh "AB mix" in a 0.5 ml pcr tube: ##90 uL IEF 40% Acrylamide (made from powder) ##10 uL Acrylamide / Bis (19:1; 38%:2%) #Using a 3-cc syringe and a 0.22 micron filter, filter ~200 uL of the ABD mix into a 1.5 mL microcentrifuge tube. #Prepare fresh 5% APS (ammonium persulfate). Notably, the APS bottle should be stored in some sort of room-temperature dessicator. We just store it inside a large plastic screw-top container. ##5 mg APS β100 uL dH20 #Prepare fresh 5% TEMED (in fume hood) ##2 uL TEMED β 38 uL dH20. #Put a few drops of 10% BSA into a 1.5 mL tube (BSA should be stored at 4'C) #Prepare the gel-casting mix (200 uL total volume). Do not add APS until immedietely prior to casting the gels. This recipe is for a 10% gel with sufficient mix for at least 8 (identical) slides. ##50 uL AB mix (FILTERED) ##4 uL 10% BSA ##138 uL dH20 ##4 uL 5% TEMED ##4 uL 5% APS #Add
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