Editing
Chris:LabNotes/SeaMicrobiome/Calendar/2015/2015-4-23
(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!
===Protocol=== *Prep tagmentation reactions (do them all separately and add enzyme last) rxn Sample Template Input 5x Tn5 Buff Water Enzyme Vol (Conc) 1 CH-S-A 10ul 38pg/ul 3ul 1ul 1ul (0.25x) 2 CH-S-A 10ul 38pg/ul 3 1 1 (0.25x * 1/2d) 3 CH-S-A 10ul 38pg/ul 3 1 1 (0.25x * 1/4d) 4 CH-S-A 10ul 38pg/ul 3 1 1 (0.25x * 1/8d) 5 CH-S-A 10ul 38pg/ul 3 1 1 (0.25x * 1/16d) 6 CH-S-A 1ul 1ng/ul 3 10 1 (0.25x) <- Pos Control 7 CH-S-A 1ul 1ng/ul 3 9 2 (0.25x) <- Verify results from previous test (rxn's 7/8 on 4/22) 8 NTC -- 3 11 1 (0.25x) <- NTC *Incubate: 55C 5min -> remove tubes and put on ice block *Add 1ul 1/100d ProtQ to each reaction *Incubate: 50C 10min -> 70C 20min -> 10C forever *Prep Exo- mastermix (add 1ul to each rxn) 1x 8.5x (ul) 10U/ul Exo- 0.1 0.85 25mM dNTP 0.4 3.4 Water 0.5 4.25 *Incubate: 37C 15min -> 75C 20min -> 10C forever *Add '''2ul i5 adaptor''' (S5xx, containing index 2) and '''2ul i7 adaptor''' (S7xx, containing index 1) in the following order. This is based off of the '''Nextera XT''' protocol recommendations for low plexity reactions found on [[File:2015_03_31_Nextera_xt_sample_preparation_guide.pdf]]. '''We also do not want to overlap adaptors from the previous libraries done on <http://genome-tech.ucsd.edu/LabNotes/index.php/Chris:LabNotes/SeaMicrobiome/Calendar/2015/2015-3-10> or the successful libraries on <http://genome-tech.ucsd.edu/LabNotes/index.php/Chris:LabNotes/SeaMicrobiome/Calendar/2015/2015-4-22>. Consequently, avoid using the combination of N701-705 & S505/506, N705/706 & S502''' rxn # Indx 1 (i7) Indx 2 (i5) 1 N701 (TAAGGCGA) S503 (TATCCTCT) 2 N702 (CGTACTAG) "" 3 N703 (AGGCAGAA) "" 4 N704 (TCCTGAGC) "" 5 N705 (GGACTCCT) "" 6 N706 (TAGGCATG) "" 7 N707 (CTCTCTAC) "" 8 (NTC) N710 "" *Add 21ul KAPA SYBR Fast (2x) to each rxn *Incubate: 72C 3min -> [98C 10sec -> 63C 30sec -> 72C 3min]x13 -> 10C *Purify using Ampure beads at 0.8:1 bead:sample ratio. Elute in 20ul H2O *Run 3ul on TBE gel *Purify again (same ratio) before sequencing if low-weight fragments appear on gel
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