Editing
Matt:LabNotes/2016-8-21
(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!
===Biotinylate RNA=== *Use Pierce RNA 3' End Biotinylation Kit (>75% ligation efficiency) *3ul of 1ug/ul UHRR #Thaw all kit components except 30% PEG and DMSO on ice #*Thaw DMSO at RT and 30% PEG at 37C for 5-10min #Adjust heating block to 85C #Transfer 3ul RNA and 1ul DMSO to 1.5ml tube and heat for 5min at 85C #Place RNA immediately on ice #Prepare labeling reaction mix by adding '''in order listed''' #*RNA volume was < 4ul due to evaporation #*Total volume < 30ul and probably had some losses when moving to 0.2ml tube because needed thermalcycler for 16C #*Use new pipette tip to mix reaction mix after PEG addition {| class="wikitable" <hiddentext>generated with [[:de:Wikipedia:Helferlein/VBA-Macro for EXCEL tableconversion]] V1.8</hiddentext> |- style="font-size:11pt" valign="bottom" | width="51" height="14" | Component | width="51" | Volume |- style="font-size:11pt" valign="bottom" | height="14" | nf H2O | align="right" | 4 |- style="font-size:11pt" valign="bottom" | height="14" | 10X RNA Ligase Buffer | align="right" | 3 |- style="font-size:11pt" valign="bottom" | height="14" | Rnase Inhibitor | align="right" | 1 |- style="font-size:11pt" valign="bottom" | height="14" | RNA (w/ 25% DMSO) | align="right" | 4 |- style="font-size:11pt" valign="bottom" | height="14" | Biotinylated Cytidine (Bis)phosphate | align="right" | 1 |- style="font-size:11pt" valign="bottom" | height="14" | T4 RNA Ligase | align="right" | 2 |- style="font-size:11pt" valign="bottom" | height="14" | 30% PEG | align="right" | 15 |- style="font-size:11pt" valign="bottom" | height="14" | Total | align="right" | 30 |} #Incubate at 16C for overnight (~15 hrs) #Add 70ul of nf-H2O #Add 100ul chloroform:isoamyl alcohol to extract RNA ligase #*96ul chloroform + 4ul isoamyl alcohol #*Vortex briefly and then centrifuge 2-3 min at high speed to separate phases #Carefully remove the top (aqueous) phase and transfer to a new tube #Add 10ul 5M NaCl, 1ul glycogen, and 300ul ice-cold 100% EtOH #Precipitate for 1hr at -20C #Centrifuge for 15min at > 13,000 x g at 4C #Remove supernatant #Wash pellet with 300ul ice-cold 70% EtOH and remove EtOH #Air-dry for ~5min #Resuspend pellet in 200ul nf-H2O
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