Editing
Matt:LabNotes/2016-6-4
(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!
==Make First Strand cDNA for in vitro Capture with TB12k_Apr2016 Probe Set== * Followed NEB M0368 protocol: [https://www.neb.com/protocols/2016/04/26/first-strand-cdna-synthesis-standard-protocol-neb-m0368] * BA8 Bulk Tissue RNA [[Matt:LabNotes/2015-5-18|from Blue]] ** 29 ng/ul ** ~4ul total * Use 1ul with random nonamer (N9) * Use 1ul with no reverse transcriptase as negative control *2 tubes {| {{table}} | align="center" style="background:#f0f0f0;"|''' Components ''' | align="center" style="background:#f0f0f0;"|''' Volume ''' |- | RNA||1 ul (30 ng) |- | N9 (100 µM)||2 ul |- | 10 mM dNTP||1 ul |- | H2O||6 ul |- | Total||10 ul |} * Denatured RNA for 5 min at 65C and then put on ice * Added the following to tubes **1ul H2O instead of ProtoScript II RT for Negative Control (to test for DNA contamination in sample) {| {{table}} | align="center" style="background:#f0f0f0;"|''' Components ''' | align="center" style="background:#f0f0f0;"|''' Volume ''' |- | 5X ProtoScript II Buffer||4 ul |- | 0.1 M DTT||2 ul |- | ProtoScript II RT (200 U/µl)||1 ul |- | RNase Inhibitor (40 U/µl)||0.2 ul |- | H2O||2.8 ul |- | Total||20 ul |} * Incubate at 25C for 5min * Incubated for one hour at 42C * Inactivated enzyme for 20 min at 65C *Stored in -20C over weekend *Purified cDNA in MinElute Qiagen PCR clean up column **Eluted 20ul
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