Editing
Chris:LabNotes/SeaMicrobiome/Calendar/2015/2015-4-9
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!
=ETOH Precipitation w/ tRNA Carrier + Library Construction Test= ==Background== *For a lot of the samples, the concentration is too low to fit into the Nextera reaction we already have established (we need ~1ng/2ul concentration). Because of this, we need to consider using ETOH precipitation in order to concentrate the samples to a level that's closer to what we need. I'm not going to use speedVac to concentrate the samples because the procedure is too variable for use. *Consequently, today I'll be testing out the ETOH precipitation and library construction protocol. Because I've already made libraries for all the Catalina Harbor samples, I'll move on to make new samples from high input North Pond samples. *The first part of the experiment will involve digesting the yeast tRNA (10mg/ml in 500ul, Life catalog #AM7119) using DNase I (from Richard, 10U/ul Roche catalog #04716728001) and then doing ethanol precipitation to ensure that all DNase is removed from the tRNA prior to adding with sample DNA ==Procedure== ===Digest tRNA with DNase I=== *Because we will need to use the tRNA for multiple samples, we will do many duplicates of this reaction and save the tRNA for later use *I followed a similar protocol as provided by Roche to digest gDNA in RNA samples (see [[File:cw_20150409_DnaseI_protocol.pdf]]) *Prep 0.2M EDTA (pH 8.0) from 0.5M EDTA by mixing: 20ul 0.5M EDTA + 30ul H2O. This will be used in the stop reaction *Mastermix Recipe: 1x 10x tRNA (10ug/ul) 5 50 10x Buff 5 50 DNase I (10U) 1 10 Water 39 390 ---------------------------- Aliquot 50ul per reaction *Incubate 37C 20min -> add 2ul 0.2M EDTA -> 75C 10min ===ETOH precipitation of digested tRNA=== *Combine 2rxn's per each non-stick tube *Add 250ul 100% ETOH, 10ul 3M NaOAc, 1.5ul glycoblue ===tRNA quantification using Qubit-RNA kit===
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