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)

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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


  1. Oil phase
    1. Start from yesterday's oil prep (Span80-4.5%, Tween20-0.1%, TritonX100-0.05% in mineral oil)
  2. Template and SYBR loading
    1. Suspend 10 uL (100 ug) beads in 100 uL wash buffer
    2. Apply magnet for 30 sec and remove supernatant
    3. Add 2 uL 10 uM template oligo per sample to bead solution; incubate at RT for 5 min
    4. Wash beads with 100 uL wash buffer; vortex to suspend; apply magnet and remove supernatant
  3. Oil/aqueous mixing (adopted from Shao/Church-adjusted for Church protocol bead concentration)
    1. Mix 95 uL TE buffer and 10 uL magnetic Dynabeads; vortex to mix
    2. Mix 20 uL aqueous into 200 uL oil mixture
    3. Vortex for 30 seconds
    4. Repeat mixing to 100 uL (5 times)
    5. Mix with vortexer for 3 minutes
    6. Dilute with pure mineral oil for imaging (1:100)