Editing
Daniel:Notebook/PosSequencing/2017-5-31
(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!
==Protocol== <ol start="8"> <li>SYBR Check</li> <ol type="A"> <li>Centrifuge sample at 13,000rpm for 1 minute</li> <li>Remove supernatant except about 10 uL</li> <li>Add 10 uL SYBR Green; incubate room temp 5 min</li> <li>Centrifuge sample at 13,000rpm for 1 minute</li> <li>Remove supernatant except about 10 uL</li> <li>Add ~40 uL 2X SSC buffer; total volume should be about 50 uL</li> <li>Add 15 uL to slide for analysis</li> </ol> ****** <li>Empty Beads SYBR Loading (Negative Control)</li> <ol type="A"> <li>Add 2 uL beads (20 ug, 20 million beads) to 100 uL wash buffer</li> <li>Pull down with magnet for 30 seconds and remove supernatant</li> <li>Add 10 uL nfH<sub>2</sub>O and 10 uL 2X SYBR mix; incubate at RT for 5 minutes</li> <li>Pull down with magnet for 30 seconds and remove supernatant</li> <li>Add 50 uL 2X SSC buffer</li> <li>Add 15 uL to slide for analysis</li> </ol> ****** <li>dsDNA SYBR Loading (Positive Control)</li> <ol type="A"> <li>Add 2 uL beads (20 ug, 20 million beads) to 100 uL wash buffer</li> <li>Pull down with magnet for 30 seconds and remove supernatant</li> <li>Add 20 uL Dynabuffer, 15 uL nfH<sub>2</sub>O, and 5 uL 500 nM barcode 2 oligo</li> <li>Incubate at RT for 15 minutes</li> <li>Pull down with magnet for 30 seconds and remove supernatant</li> <li>Wash with 100 uL dynabuffer</li> <li>Pull down with magnet for 30 seconds and remove supernatant</li> <li>Add 10 uL nfH<sub>2</sub>O and 10 uL 2X SYBR mix; incubate at RT for 5 minutes</li> <li>Pull down with magnet for 30 seconds and remove supernatant</li> <li>Add 50 uL 2X SSC buffer</li> <li>Add 15 uL to slide for analysis</li> </ol>
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