Editing
Andrew:Notebook/C1 InTube 150525
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!
==Troubleshooting HLS-Tn5 protocol== * AR150527_Tn5_tubeTest ===Motivation=== * My last experiment tested HLS+ProtQ, Tn5-059, ProtQ digestion, and PCR with standard Nextera XT v2 barcoded primers. * The results were negative: [[http://genome-tech.ucsd.edu/LabNotes/index.php/Andrew:Notebook/C1_OpenApp_150429 Troubleshooting the Tn5 titration experiment]] * This indicates that either the protease or Tn5 died, so I am going to make fresh aliquots and repeat. * I will also include gDNA from ~350 [http://genome-tech.ucsd.edu/LabNotes/index.php/Arichard:Samples/cells150514 GM12878] cells extracted with HLS + G-HCl and purified with Ampure XP. * I will use both standard barcoded primers as well as the Nextera transposon sequences. * If the transposon sequences work, I can do the Tn5 titration on chip with a back-loaded Tn5 curve and common PCR mix, followed by barcoded 2nd PCR off-chip. ===Samples=== * 6 samples total, 3 samples per protocol * The products from reactions 1-3 will serve as template for reactions 4-6, similar to the C1 protocol {| {{table}} | align="center" style="background:#f0f0f0;"|'''Condition''' | align="center" style="background:#f0f0f0;"|'''1''' | align="center" style="background:#f0f0f0;"|'''2''' | align="center" style="background:#f0f0f0;"|'''3''' | align="center" style="background:#f0f0f0;"|'''4''' | align="center" style="background:#f0f0f0;"|'''5''' | align="center" style="background:#f0f0f0;"|'''6''' |- | Protocol||Transposon PCR||Transposon PCR||Transposon PCR||Barcode 2nd PCR||Barcode 2nd PCR||Barcode 2nd PCR |- | Sample||350 GM12878||Purified DNA||NTC||350 GM12878||Purified DNA||NTC |- | |} ===Protocol=== * I got a fresh aliquot of 20 mg/mL Qiagen Protease from Brandon. * Adjustment factors: ** HLS ProtQ mix --> 1.5X ** Tn5 --> 1.75X ** ProtQ --> 1.2X ** PCR --> 2.2X Transposon primed 1st PCR # Start with 1 uL: Cells, DNA, or NTC # Make HLS ProtQ mix for 5 reactions, 2 uL each, 1.5X: 10 uL of 7.5 mg/mL ProtQ, 45 mM Tris-HCl ## 3.75 uL 20 mg/mL ProtQ ## 1.125 uL 400 mM Tris-HCl ## 5.125 uL H2O ## Longer lysis and heat-kill: 30 min at 50 C and 30 min at 70 C. # Make Tn5 mix for 4 reactions, 4 uL each, 1.75X: 16 uL of 0.15X Tn5 in Tn5 diluent ## 2.4 uL 1X Tn5 ## 13.6 uL Tn5 diluent # Make ProtQ mix for 4 reactions, 30 uL each, 1.2X: 120 uL of 0.24 mg/mL ProtQ, 30 mM Tris-HCl ## 1.44 uL 20 mg/mL ProtQ ## 9 uL 400 mM Tris-HCl ## 109.5 uL H2O # Make PCR mix for 4 reactions, 30 uL each, 2.2X: 120 uL of 2.2X NPM, 440 nM each primer ## 79 uL 3.33X NPM ## 5.28 uL 10 uM Nextera P7 transposon ## 5.28 uL 10 uM Nextera P5 transposon ## 30.44 uL H2O * Used 68 degree annealing temp Barcoded 2nd PCR # 3 uL 1st PCR product # 1 uL 10 uM N7 primer # 1 uL 10 uM S5 primer # 5 uL 2X KAPA SYBR Fast ===Gels=== [[File:AR150527_gels_Slide1.jpg|600px]] [[File:AR150527_gels_Slide2.jpg|600px]]
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)
Template used on this page:
Template:Table
(
edit
)
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