Editing
Daniel:Notebook/PosSequencing/2017-4-11
(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!
=Emulsion Test-Variables= [[Daniel:Notebook/PosSequencing|Back to Calendar]] The purpose of today is to get a feel of the protocols I have with the emulsion making. I'll be using [[Daniel:Notebook/PosSequencing/2017-4-10|yesterday]]'s results as a positive and a starting point. As such I'll be testing each of the components in the experiment from yesterday, stretching them both in larger and smaller concentration. I'll want to measure both droplet size and '''bead loading'''. See below for the bead loading, which will be the more important of the two variables. ==Bead Loading Test== <ol> <li>Oil phase</li> <ol type="A"> <li>Start from [[Daniel:Notebook/PosSequencing/2017-4-10|yesterday]]'s oil prep (Span80-4.5%, Tween20-0.1%, TritonX100-0.05% in mineral oil)</li></ol> <li>Oil/aqueous mixing (adopted from Shao/Church)</li> <ol type="A"> <li>Mix 95 uL TE buffer and 5 uL magnetic beads; vortex to mix</li> <li>Mix 20 uL aqueous into 200 uL oil mixture</li> <li>Vortex for 30 seconds</li> <li>Repeat mixing to 100 uL (5 times)</li> <li>Mix with vortexer for 3 minutes</li> <li>Dilute with pure mineral oil for imaging (1:100)</li> </ol> <li>Oil/aqueous mixing (adopted from Shao/Church-adjusted for Church protocol bead concentration)</li> <ol type="A"> <li>Mix 95 uL TE buffer and 10 uL magnetic [https://www.thermofisher.com/order/catalog/product/65001 Dynabeads]; vortex to mix</li> <li>Mix 20 uL aqueous into 200 uL oil mixture</li> <li>Vortex for 30 seconds</li> <li>Repeat mixing to 100 uL (5 times)</li> <li>Mix with vortexer for 3 minutes</li> <li>Dilute with pure mineral oil for imaging (1:100)</li> </ol> </ol> ===NEB Beads Results=== <gallery perrow=3 heights=300px widths=300px> File:20170411-B1Emulsion-BF2.jpg|Brightfield image-Field 1 File:20170411-B1Emulsion-CFP2.jpg|CFP image-Field 1 File:20170411-B1Emulsion-BF4.jpg|Brightfield image-Field 2 File:20170411-B1Emulsion-CFP4.jpg|CFP image-Field 2 File:20170411-B1Emulsion-DAPI4.jpg|DAPI image-Field 2 </gallery> The results here are kind of mixed. Basically, The beads seem sparse and its hard to tell what are beads and what are bubbles. Field 2 shows a good example of something that may be a bead. However, I think the autofluorescence is pretty low for these beads. It does appear in both DAPI and CY5 channels (which is encouraging). However, there just aren't a lot of beads compared to what I think are bubbles, if that is the case. [[Category:PosSeq]] [[Category:20170411]] ===Dynabeads Results=== I definitely feel more confident here, but it still isn't easy. <gallery perrow=3 heights=300px widths=300px> File:20170411-D1Emulsion-BF2.jpg|Field 1-Brightfield File:20170411-D1Emulsion-DAPI2.jpg|Field 1-Dapi File:20170411-D1Emulsion-Overlay2.jpg|Field 1-Overlay File:20170411-D1Emulsion-BF3.jpg|Field 2-Brightfield File:20170411-D1Emulsion-DAPI3.jpg|Field 2-Dapi File:20170411-D1Emulsion-Overlay3.jpg|Field 2-Overlay </gallery>
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