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!
=Adjusted Tn5 Rxn Volume Test II= ==Background== *Yesterday, I tried adjusting the Tn5 reaction volume to 15ul (see <http://genome-tech.ucsd.edu/LabNotes/index.php/Chris:LabNotes/SeaMicrobiome/Calendar/2015/2015-4-22>). However, only reactions 5&6 showed good smears (with rxn's 7&8 being very dim). This means that the 1ul 0.25x supermutant Tn5059 was sufficient for digesting 1ng of DNA but when we increased that to 2ul 0.25x Tn5059, there was over-digestion. Consequently, it looks like for the ~400pg DNA template input, I likewise over-digested the template leading to no product on the gel. *Today, I'll be working on another test, this time trying out different enzyme concentration inputs in order to lower the concentration to get some product. *In order to not run out of template, I'll be reverting back to the Catalina Harbor samples (particularly testing with CH-S-A, which was 63.2ng/ul). Consequently, I'll be running the following test: rxn Sample Template Input Enzyme Conc (1ul added) 1 CH-S-A 10ul 38pg/ul 0.25x 2 CH-S-A 10ul 38pg/ul 0.25x * 1/2d 3 CH-S-A 10ul 38pg/ul 0.25x * 1/4d 4 CH-S-A 10ul 38pg/ul 0.25x * 1/8d 5 CH-S-A 10ul 38pg/ul 0.25x * 1/16d 6 CH-S-A 1ul 1ng/ul 0.25x <- Pos Control 7 CH-S-A 1ul 1ng/ul 2ul 0.25x <- Verify results from previous test (rxn's 7/8 on 4/22) 8 NTC -- 0.25x <- NTC ==Procedure== ===Sample Dilution=== *We want to dilute the samples to 1ng/ul using the following dilutions. These will be utilized for our positive control when performing library construction on the supermutant (we will be utilizing exactly 1ul of the 1ng/ul sample for the positive control) Concentration ul Sample ul Water CH-S-A 63.2ng/ul 1ul 62.2ul *In order to make the concentration more representative of the lower concentrated South Pacific Gyre samples, we will further dilute to 38pg/ul (0.038ng/ul) using the following dilutions: ('''Note: We are utilizing the 1ng/ul dilution fo the sample and further diluting it down to 38pg/ul''') Concentration ul Sample ul Water CH-S-A 1ng/ul 1 25.32 ===Prepare Enzyme Dilutions=== *We want to dilute the supermutant-Tn5 ('''This was already done previously on the 3/31/2015 library construction experiment and we will use the same diluted enzymes''') **supermutant-Tn5 provided in 25x concentration, so dilute using the following: 0.5ulA (Tn5059 ME-A Tsm 25x) + 0.5ulB (Tn5059 ME-B Tsm 25x) + 99ul Tn5 standard storage buffer Tsm diluent *We want to further dilute the enzyme from 0.25x with the following serial dilution: Starting Conc ul Tn5 ul Water Ending Conc 0.25x 2 2 0.25x * 1/2d 0.25x * 1/2d 2 2 0.25x * 1/4d 0.25x * 1/4d 2 2 0.25x * 1/8d 0.25x * 1/8d 2 2 0.25x * 1/16d *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 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 ==Results== *Below is the gel image from this experiment (x, Low Mass Ladder, x, rxn's 1->8) [[File:cw_ZhangLab_2_2015-04-23_21hr_11min_ntx_volume_test_2.jpg|500px]]
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