Editing
Dinh:5hmC Lambda Controls Protocol
(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!
== Preparation of the sequencing and digestion spike-in controls== * Design primers to amplify three different regions in Lambda DNA: * All primers can amplify DNA at 55C with good efficiency. <nowiki>Lambda_B_T_F TAGGAAGATAGTGTTTATGT Lambda_B_T_R CCAACAAAAATTTCTCCTAT Lambda_B_C_F CAGGAAGACAGTGCTCATGC Lambda_B_C_R CCAGCAGGGATTTCTCCTGT Lambda_E_T_F TGTTATTTATGTTGTATGGTGT Lambda_E_T_R CAACTAACTTCTTTTCTTTCAC Lambda_E_C_F TGTTATTCATGTTGCATGGTGC Lambda_E_C_R CAGCTGACTTCTTTTCTTTCAC Lambda_F_T_F TTGTTTATAGGAGATATGGTAGA Lambda_F_T_R CTTACTAACCAATTCCTAAA Lambda_F_C_F ttgctcataggagatatggt Lambda_F_C_R cttgctaaccaattcctagg </nowiki> * Use the "C" primers to amplify lambda DNA with ZymoTaq polymerase * Perform size selection of the amplicons. IMPORTANT to prevent trace amount of unmodified DNA to remain in the mixtures. * Use size selected amplicons with different dNTP mixtures containing: dCTPs, dmCTPs, or dhmCTPs. * Reaction set up: Reagent Volume per reaction Reaction buffer 25 uL dNTPs mixture 5 uL (for 5hmC mixture (2.5mM), and 1.25 uL for C/mC mixture (10 mM) Primers (F/R, 10uM) 2 uL Template (10 nM) 1 uL Nuclease-free water to 50 uL Zymo Taq Polymerase 0.4 uL * Program: 95C (10min), (95C 30s -> 55C 30s -> 72C 1 min)x30, 72C 7 min * Purify each mixture with PCR cleanup columns and quantify with Qubit. B - 5hmC 27.5 ng/uL E - 5mC 25.6 ng/uL F - C 14.4 ng/uL
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