Daniel:Notebook/PosSequencing/2017-4-12: Difference between revisions
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==Bead Loading Test== | ==Bead Loading Test== | ||
'''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> | ||
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<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 wash buffer </li> | |||
<li>Apply magnet for 30 sec and remove supernatant</li> | |||
<li>Add 2 uL 10 uM template oligo per sample to bead solution; incubate at RT for 5 min</li> | |||
<li>Wash beads with 100 uL wash buffer; vortex to suspend; apply magnet and remove supernatant</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"> |
Revision as of 20:17, 12 April 2017
Emulsion Test-Variables (Started Yesterday)
Bead Loading Test
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 wash buffer
- Apply magnet for 30 sec and remove supernatant
- Add 2 uL 10 uM template oligo per sample to bead solution; incubate at RT for 5 min
- Wash beads with 100 uL wash buffer; vortex to suspend; apply magnet and remove supernatant
- 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)