Editing
Sam:LabNotes/Microbiome-new/2010-7-25
(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!
='''Extra-library amplification using rt-PCR'''= *Since there are 10uL template left from the 1st step of Nextera library construction. Only 5uL were used for PCR amplification. *I am trying to use the other 5uL template(transposon tagged DNA) for amplification. *Set up the reaction on cold block in EP-white PCR-strip tubes: 1rxn x2.2 rxn RT-PCR grade H2O 16.8 36.96 DNA lib (from last step) 5.0 - 2X Nextera PCR buf. 25.0 55.00 50X Nextera primier cocktail 1.0 2.20 - Illumina-compatible 50X Nextera adaptor 2 1.0 2.20 - could be stwitch to barcoded library (optional) Netera PCR enzyme 1.0 2.20 50X SYBR-green I 0.2 0.44 ------------------------------------------- 50.0 99.00/2.2=45 --- template 5uL '''NOTE: I use the "no.2" SYBR-green 50X''' Stop the reaction before the amplification curve reach pleatue ==Results== *3uL amplified libraries were validated using 6%TBE-PAGE with standard long staining method(200V, 30min). [[File:sam072910-nextera lib amp-2nd amp.jpg|300px]] *The 90min-library showed good range between 200-400bp *The 10hr-library is probably over-loaded on gel. I need to do the nanodrop on this amplicon and validate again. **After AMpure amplification the concentration could be raised up becasued the totoal volume was changed from 50uL to 30uL (I used 30uL H2O to elute the purified DNA). *The comparison of before and after AMpure bead purification showed no significatn loss with the purification method.
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