Editing
Noi/NOTES/2014-3-26
(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!
==== Removal of amplification adaptors ==== * I reduce digestion volume to 120ul as I reduce DNA amount to ~10ug <u>'''1x rxn'''</u> <u>'''2.5x rxn'''</u> ssDNA (85.3ng/ul) 60ul 150ul (DNA amount /tube = 5.11ug) USER 5ul 12.5ul H2O 15ul 37.5ul -------------------------------------------------------- Total 80ul Aliquot 80ulx2 and 40ulx1 : - Incubate at 37C for 2h <u>'''1x rxn'''</u> <u>'''2.6x rxn'''</u> 10x DpnII buffer 10ul 26ul 100uM RE-DpnII-V6 guide oligos 5ul 13ul -------------------------------------------------------- Total 15ul : - Add 15ul and 7.5ul of 10X DpnII buffer and RE-DpnII-V6 guide oligos to 80ul and 40ul reaction tubes, respectively. : - Mix well and incubate at 94C for 3min -> 37 C 3min : - Add 5ul or 2.5ul of DpnII to 80ul and 40ul reaction tubes, respectively. : - Mix by pipetting 10 times on thermocycler : - Incubate at 37C for 3h. Heat inactivate at 75C for 20min : - Purify USER/DpnII digested ssamplicon with 3x ssDNA/RNA clean & concentrator column, ZymoResearch : - Follow protocol provided with the kit (for volume 120ul) : - Elute each column with 62ul H2O. Total volume left = 180ul) : - N.D. ssDNA = 44.5ng/ul : - Yields = 180ul * 44.5ng/ul = 8ug
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