Editing
Noi/NOTES/2014-10-22
(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 WGBS of cfDNA from plasma using NEXTflex Cell Free DNA Seq Kit = * The kit just arrived today, and I want to finish experiment ASAP. I then continue to do experiment using the same samples as of KAPA Hyper Prep kit * I set up the reaction similar to KAPA Hyper Prep kit by reducing total volume/amount of reagent in each step to a half of instruction of the kit == Bisulfite conversion == * I performed bisulfite conversion using the same procedure following manufacturer's instruction of '''EZ DNA Methylation-Lightning kit''' and elute with 34ul elution buffer. '''''<span style="color:crimson"><u>Prep</u></span>'''''<br> :- Aliquot 6.12mL of binding buffer & mix with 5.2ul of 10ng/ul tRNA :- Aliquot 5.1mL of Washing buffer :- Aliquot 2.1mL of Desulfonation buffer :- Aliquot 340ul of Elution buffer == Amplification == * For amplification, I can not use KAPA HIFI HotStart Ready Mix since I do bisulfite conversion on the DNA. I will use Pfu Turbo Cx for amplification. * I normally do 2 round of PCR for the input less than 10ng. In this experiment, I will do a quick test single round PCR in small volume and run PCR for 18 & 20 cycles. The kit recommend 17-19 cycles for 1ng input of normal sheared gDNA. I will increase few more cycles for bisulfite converted DNA. '''Adapted PCR set up in 50ul''' '''Components 1x rxn''' 10X Reaction Buffer 5.00 10mM dNTP mix 1.25 10X Lib Amp Primer mix 5.00 Adaptor ligated library 30.00 Pfu Turbo Cx 1.00 H2O 7.75 Total 50.00 * For the quick test, I will use a little less of DNA template
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