Editing
Chris:LabNotes/SeaMicrobiome/Calendar/2015/2015-6-17
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!
=Nextera Lib Construction on Catalina Harbor Samples (Ez-Tn5)= ==Background== *Previously, I used the supermutant-Tn5 in order to prepare libraries for all Catalina harbor samples (see <http://genome-tech.ucsd.edu/LabNotes/index.php/Chris:LabNotes/SeaMicrobiome/Calendar/2015/2015-3-31>). However, we want to do library construction using only Ez-Tn5 as per Karsten's request. According to previous tests, an enzyme concentration of 0.25x worked well for 1ng of input *We want to dilute the samples to ~1ng/ul using the following dilutions: (we will use the same dilutions as done previously for the sm-Tn5059 library construction) Concentration ul Sample ul Water Test 1 5.22ng/ul 1 4.22 Test 2 5.09 1 4.09 CH-P-A 6.67 1 5.67 CH-P-B 18.8 1 17.8 CH-P-C 14.1 1 13.1 CH-P-D 13.6 1 12.6 CH-O-A (Filter 1) 12.6 1 11.6 CH-O-B (Filter 2) 27.6 1 26.6 *The necessary reagents can be found in one of four places: **Andrew's 4C box **-20C 2nd shelf in Midas -20C box **-20C 2nd shelf in Nextera primer box **-20C 3rd shelf Brandon's Tn5 box (contains the EZ-Tn5 and supermutant) *Below are the reactions we'll be using today: Rxn # Template Enzyme Enzyme Dilution 1 Test 1 EZ-Tn5 0.25x 2 Test 2 "" "" 3 CH-P-A "" "" 4 CH-P-B "" "" 5 CH-P-C "" "" 6 CH-P-D "" "" 7 CH-O-A "" "" 8 CH-O-B "" "" 9 --(NTC) "" "" ==Procedure== ===Prepare Enzyme Dilutions=== *We want to dilute the EZ-Tn5 **EZ-Tn5 provided in 5x concentration, so dilute using the following: 0.25ulA + 0.25ulB + 9.5ul Tn5 standard storage buffer Tsm diluent *We also want to dilute ProtQ 1/100 from 20mg/ml. In order to do this, we add 2ul ProtQ + 198ul Water ===Protocol=== *Prep tagmentation mastermix 1x 9.5x (ul) Template 1 -- Nxt 1 9.5 <- EZ-Tn5 HMW Buff 1 9.5 <- renamed "5x Tn5 Buffer" Water 2 19 *Incubate: 55C 5min -> remove tubes and put on ice block *Add 1 ul 1/100d ProtQ to each reaction *Incubate: 50C 10min -> 70C 20min -> 10C forever *Prep Exo- mastermix (add 1ul to each rxn) 1x 9.5x (ul) 10U/ul Exo- 0.1 .95 25mM dNTP 0.4 3.8 Water 0.5 4.75 *Incubate: 37C 15min -> 75C 20min -> 10C forever *Add the following i5 adaptor (S5xx, containing index 2) and 1ul i7 adaptor (N7xx, 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 <http://genome-tech.ucsd.edu/LabNotes/index.php/Chris:LabNotes/SeaMicrobiome/Calendar/2015/2015-3-31>. Consequently, avoid using the combination of N701-705 and S505/506/S502''' rxn # Indx 1 (i7) Indx 2 (i5) 1 N701 (TAAGGCGA) S503 (CTCTCTAT) 2 N702 (CGTACTAG) "" 3 N703 (AGGCAGAA) "" 4 N704 (TCCTGAGC) "" 5 N705 (GGACTCCT) "" 6 N706 (TAGGCATG) "" 7 N707 (CTCTCTAC) "" 8 N710 (CGAGGCTG) "" 9 (NTC) N711 "" *Prep PCR mastermix -> add 11ul to each rxn 1x 10.5x KAPA SYBR Fast (2x) 10 105 Water 1 10.5 *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