Editing
Dinh/Dinh 2014/NOTES/2014-6-23
(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!
== September 10, 2014 update == *Antarctic Phosphatase to prevent self-circularization of vector (Found some Antarctic Phosphatase in the freezer) Reaction: 100 ng of pUC19 vector ( 1.3 uL x 79.2 ng/uL ) 1x Antarctic Phosphatase Reaction buffer ( 1.0 uL x 10x bufer) 5 units of Antarctic Phosphatase ( 1.0 uL x 5 units/uL) 6.7 uL of water Program: 15 minutes at 37C 5 minutes at 70C to heat inactivate *Ligation Reaction: 100 ng of pUC19 vector ( 10 uL of phosphatase reaction ) 188 ng of insert ( 6.0 uL x 30.0 ng/uL ) 1x Ligase buffer ( 5.0 uL x 10x buffer) 600 units T4 DNA Ligase ( 1.5 uL x 400 unit/uL) 27.5 uL of water Program: Room temperature for at least 10 minutes (cohesive sticky ends) *Transformation into Top10 E. coli Used all 50 uL ligation reaction to transform 1 tube of competent cells. Incubate on ice for 30 minutes Heatshock 1 minute at 42C Add 250 uL SOC media Incubate on shaker at 37C for 1.5 hr. Spread all of product onto plate Incubate at 37C overnight *Purify Stoffel & Taq
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