Daniel:Notebook/PosSequencing/2017-4-11: Difference between revisions
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<li>Oil phase</li> | <li>Oil phase</li> | ||
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<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> | <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> | <li>Oil/aqueous mixing (adopted from Shao/Church)</li> | ||
<ol type="A"> | <ol type="A"> | ||
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<li>Dilute with pure mineral oil for imaging (1:100)</li> | <li>Dilute with pure mineral oil for imaging (1:100)</li> | ||
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<li>Oil/aqueous mixing (adopted from Shao/Church-adjusted for Church protocol bead concentration)</li> | |||
<li>Oil/aqueous mixing (adopted from Shao/Church)</li> | |||
<ol type="A"> | <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 95 uL TE buffer and 10 uL magnetic [https://www.thermofisher.com/order/catalog/product/65001 Dynabeads]; vortex to mix</li> | ||
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</ol> | </ol> | ||
</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> |
Latest revision as of 16:13, 19 April 2017
Emulsion Test-Variables[edit]
The purpose of today is to get a feel of the protocols I have with the emulsion making. I'll be using 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[edit]
- Oil phase
- Start from yesterday's oil prep (Span80-4.5%, Tween20-0.1%, TritonX100-0.05% in mineral oil)
- Oil/aqueous mixing (adopted from Shao/Church)
- Mix 95 uL TE buffer and 5 uL magnetic beads; vortex to mix
- Mix 20 uL aqueous into 200 uL oil mixture
- Vortex for 30 seconds
- Repeat mixing to 100 uL (5 times)
- Mix with vortexer for 3 minutes
- Dilute with pure mineral oil for imaging (1:100)
- Oil/aqueous mixing (adopted from Shao/Church-adjusted for Church protocol bead concentration)
- Mix 95 uL TE buffer and 10 uL magnetic Dynabeads; vortex to mix
- Mix 20 uL aqueous into 200 uL oil mixture
- Vortex for 30 seconds
- Repeat mixing to 100 uL (5 times)
- Mix with vortexer for 3 minutes
- Dilute with pure mineral oil for imaging (1:100)
NEB Beads Results[edit]
- 20170411-B1Emulsion-BF2.jpg
Brightfield image-Field 1
- 20170411-B1Emulsion-CFP2.jpg
CFP image-Field 1
- 20170411-B1Emulsion-BF4.jpg
Brightfield image-Field 2
- 20170411-B1Emulsion-CFP4.jpg
CFP image-Field 2
- 20170411-B1Emulsion-DAPI4.jpg
DAPI image-Field 2
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.
Dynabeads Results[edit]
I definitely feel more confident here, but it still isn't easy.
- 20170411-D1Emulsion-BF2.jpg
Field 1-Brightfield
- 20170411-D1Emulsion-DAPI2.jpg
Field 1-Dapi
- 20170411-D1Emulsion-Overlay2.jpg
Field 1-Overlay
- 20170411-D1Emulsion-BF3.jpg
Field 2-Brightfield
- 20170411-D1Emulsion-DAPI3.jpg
Field 2-Dapi
- 20170411-D1Emulsion-Overlay3.jpg
Field 2-Overlay