Editing
Noi/NOTES/2013-4-30
(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!
== Ligation reaction mix == {| class="wikitable" style="text-align:center; | align="center" style="background:#f0f0f0;"|'''Ligation reaction mix''' | align="center" style="background:#f0f0f0;"|'''1x rxn''' | align="center" style="background:#f0f0f0;"|'''9x rxn mix''' |- | KAPA 5X Ligation buffer||10.00||90.00 |- | KAPA DNA ligase||5.00||45.00 |- | H2O||19.33||173.97 |- | Total||34.33||308.97 |} * Aliquot 34.33 ul of ligation reaction mix to each tube * Transfer 4ul of sample to reaction tubes * As I mentioned before, if the sample is already in a clear tube, just add ligation reaction mix directly to fragmented DNA and adjust volume in ligation reaction mix ** I used 0.2 ml PCR Tube Strips, low-Profile tube (Biorad) to set up reaction * Rinse the tube with 10ul H2O and transfer all to reaction tube (total volume now is 48.33ul) * Add 1.67ul of 1.5uM loop adaptor * 20C for 15min (no shaking) * Add 1.5ul USER (come with the kit), 37C for 15min ** For the library prep using NEBNext® DNA Library and this loop adaptor, they used 3ul USER in total volume 50ul or 83.5ul, 37 C for 15 min. I used a little less, so might increase volume of USER to 2ul/ reaction or use 1.5ul with longer incubation (~30min) for the completion of USER digestion * Bead purification: ** 50ul AmPure beads mix by pipetting 10 times ** Wait 10min (no shaking) ** Sit on magnetic stand 5min ** Wash twice with freshly prepared 80% EtOH (after adding 80% EtOH, I wait for 30sec before pipette up and down few times and discard spnt) *** I wash with 160ul of 80% EtOH since I used low profile tube. ** Dry the beads for 3 min * Eluted with 20ul H2O (resuspend and wait for 2min, used all for PCR)
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