Editing
Daniel:Protocols/CProbeLigate
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!
=C Probe Hybridization= [[Daniel Jacobsen|Back to Notebook]] This page describes the protocol for hybridizing C probes as used in PLAYR Design (as from [http://www.nature.com/nmeth/journal/v13/n3/abs/nmeth.3742.html Nolan lab paper]). ==Protocol== Resuspend oligos in nuclease free water to concentration of 100 uM <ol> <li>Fix cells</li> <ol type="A"> <li>Pellet cells at 600g for 3 min and resuspend at concentration of ~1,000,000/mL</li> <li>Fix cells in medium with 1.6% paraformaldehyde for 10 min at RT</li> <li>Wash cells with wash buffer</li> <li>Pellet cells and permeabilize with ice-cold methanol for 10 min on ice</li> Once in methanol cells may be stored long term </ol> <li>Hybridization</li> <ol type="A"> <li>Pellet cells via centrifugation at 600g for 3 min</li> <li>Heat C probes to 90C for 5 min; Chill probes on ice to quench</li> <li>Add probes to cells for final concentration of 100 nM</li> <li>Incubate probes at 40C for 1 hour with vigorous agitation</li> <li>Wash three times with wash buffer and pelleting at 600xg for 3 min</li> <li>Incubate cells for 20 min at 40C in stringent wash buffer</li> </ol> </ol> ==Buffers== '''Wash Buffer''' *PBS *0.1% Tween *4 U/mL RNasin '''Stringent Wash Buffer''' *PBS *4X SSC *40 U/mL RNasin '''Hybridization Buffer''' *1X SSC *2.5% vol/vol polyvinylsulfonic acid *20 mM ribonucleoside vanadyl complex *40 U/mL RNasin *0.1% Tween *100 ug/mL salmon sperm
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