Daniel:Notebook/PosSequencing/2017-4-12: Difference between revisions
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==Bead Loading Test== | ==Bead Loading Test== | ||
[https://www.thermofisher.com/us/en/home/references/protocols/proteins-expression-isolation-and-analysis/protein-isolation-protocol/dynabeads-myone-streptavidin-c1.html#prot4 Recommended info on Dynabeads] | |||
'''Dynabeads Buffer Prep''' | |||
{| class="wikitable" <hiddentext>generated with [[:de:Wikipedia:Helferlein/VBA-Macro for EXCEL tableconversion]] V1.8</hiddentext> | |||
|- style="background-color:#C4D79B;font-size:12pt;font-weight:bold" align="center" | |||
| width="105" height="30" | Reagent | |||
| width="85" | Stock | |||
| width="85" | Final | |||
| width="85" | Dilution | |||
| width="85" | Amt in 10 mL | |||
|- style="font-size:12pt" | |||
|style="font-weight:bold" height="15" valign="bottom" | NaCl | |||
| align="center" valign="bottom" | 5 M | |||
| align="center" valign="bottom" | 2 M | |||
| align="center" align="center" valign="bottom" | 2.5 | |||
| align="center" valign="bottom" | 4 mL | |||
|- style="background-color:#D9D9D9;font-size:12pt" | |||
|style="font-weight:bold" height="15" valign="bottom" | Tris-HCl | |||
| align="center" valign="bottom" | 500 mM | |||
| align="center" valign="bottom" | 10 mM | |||
| align="center" align="center" valign="bottom" | 50 | |||
| align="center" valign="bottom" | 200 uL | |||
|- style="font-size:12pt" | |||
|style="font-weight:bold" height="15" valign="bottom" | EDTA | |||
| align="center" valign="bottom" | 0.5M | |||
| align="center" valign="bottom" | 1 mM | |||
| align="center" align="center" valign="bottom" | 500 | |||
| align="center" valign="bottom" | 20 uL | |||
|- style="background-color:#D9D9D9;font-size:12pt" | |||
|style="font-weight:bold" height="15" valign="bottom" | nf H2O | |||
| align="center" valign="bottom" | NA | |||
| align="center" valign="bottom" | NA | |||
| align="center" valign="bottom" | NA | |||
| align="center" valign="bottom" | 5.8 mL | |||
|} | |||
<ol> | <ol> | ||
Line 9: | Line 53: | ||
<ol type="A"> | <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>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>Template and SYBR loading</li> | |||
<ol type="A"> | |||
<li>Suspend 10 uL (100 ug) beads in 100 uL Dynabuffer</li> | |||
<li>Apply magnet for 30 sec and remove supernatant</li> | |||
<li>Suspend 10 uL (100 ug) beads in 20 uL Dynabuffer (conc. 5 ug/uL)</li> | |||
<li>Add 20 uL 10 uM template oligo per sample to bead solution; incubate at RT for 15 min</li> | |||
<li>Wash beads with 100 uL wash buffer; vortex to suspend; apply magnet and remove supernatant</li> | |||
<li>Add 100 uL of Wash buffer with 1 uL of 100:1 diluted SYBR gold (Final dilution 1:10000)</li> | |||
<li>Wash beads with 100 uL wash buffer; vortex to suspend; apply magnet and remove supernatant</li> | |||
<li>Repeat wash step 2X more</li> | |||
<li>Resuspend beads in 100 uL TE buffer</li> | |||
</ol> | |||
<li>Oil/aqueous mixing (adopted from Shao/Church-adjusted for Church protocol bead concentration)</li> | <li>Oil/aqueous mixing (adopted from Shao/Church-adjusted for Church protocol bead concentration)</li> | ||
<ol type="A"> | <ol type="A"> | ||
<li>Mix 20 uL aqueous into 200 uL oil mixture</li> | <li>Mix 20 uL aqueous into 200 uL oil mixture</li> | ||
<li>Vortex for 30 seconds</li> | <li>Vortex for 30 seconds</li> | ||
Line 19: | Line 74: | ||
</ol> | </ol> | ||
</ol> | </ol> | ||
===Results=== | |||
The Dapi channel (and all other fluorescent channels) shows an annoying level of background, which unfortunately follows a gradient. Notice the left to right gradient with high background on the left and low background on the right. This is a result of the shutter, but can't be fully eliminated by changing the shutter settings. This gradient is actually the best since it is easy to see and can be accounted for accordingly. | |||
<gallery perrow=3 heights=300px widths=300px> | |||
File:20170412-D1SYBREmulsion-BF3.jpg|Field 1-Brightfield | |||
File:20170412-D1SYBREmulsion-DAPI3.jpg|Field 1-Dapi | |||
File:20170412-D1SYBREmulsion-Overlay3.jpg|Field 1-Overlay | |||
File:20170412-D1SYBREmulsion-BF5.jpg|Field 2-Brightfield | |||
File:20170412-D1SYBREmulsion-DAPI5.jpg|Field 2-Dapi | |||
File:20170412-D1SYBREmulsion-Overlay5.jpg|Field 2-Overlay | |||
</gallery> | |||
[[Category:PosSeq]] [[Category:20170411]] |
Latest revision as of 16:13, 19 April 2017
Emulsion Test-Variables (Started Yesterday)[edit]
Bead Loading Test[edit]
Dynabeads Buffer Prep
Reagent | Stock | Final | Dilution | Amt in 10 mL |
NaCl | 5 M | 2 M | 2.5 | 4 mL |
Tris-HCl | 500 mM | 10 mM | 50 | 200 uL |
EDTA | 0.5M | 1 mM | 500 | 20 uL |
nf H2O | NA | NA | NA | 5.8 mL |
- Oil phase
- Start from yesterday's oil prep (Span80-4.5%, Tween20-0.1%, TritonX100-0.05% in mineral oil)
- Template and SYBR loading
- Suspend 10 uL (100 ug) beads in 100 uL Dynabuffer
- Apply magnet for 30 sec and remove supernatant
- Suspend 10 uL (100 ug) beads in 20 uL Dynabuffer (conc. 5 ug/uL)
- Add 20 uL 10 uM template oligo per sample to bead solution; incubate at RT for 15 min
- Wash beads with 100 uL wash buffer; vortex to suspend; apply magnet and remove supernatant
- Add 100 uL of Wash buffer with 1 uL of 100:1 diluted SYBR gold (Final dilution 1:10000)
- Wash beads with 100 uL wash buffer; vortex to suspend; apply magnet and remove supernatant
- Repeat wash step 2X more
- Resuspend beads in 100 uL TE buffer
- Oil/aqueous mixing (adopted from Shao/Church-adjusted for Church protocol bead concentration)
- 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)
Results[edit]
The Dapi channel (and all other fluorescent channels) shows an annoying level of background, which unfortunately follows a gradient. Notice the left to right gradient with high background on the left and low background on the right. This is a result of the shutter, but can't be fully eliminated by changing the shutter settings. This gradient is actually the best since it is easy to see and can be accounted for accordingly.
- 20170412-D1SYBREmulsion-BF3.jpg
Field 1-Brightfield
- 20170412-D1SYBREmulsion-DAPI3.jpg
Field 1-Dapi
- 20170412-D1SYBREmulsion-Overlay3.jpg
Field 1-Overlay
- 20170412-D1SYBREmulsion-BF5.jpg
Field 2-Brightfield
- 20170412-D1SYBREmulsion-DAPI5.jpg
Field 2-Dapi
- 20170412-D1SYBREmulsion-Overlay5.jpg
Field 2-Overlay