Daniel:Notebook/PosSequencing/2017-4-12: Difference between revisions

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>Djacobse
>Djacobse
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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'''
'''Dynabeads Buffer Prep'''
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<li>Template and SYBR loading</li>
<li>Template and SYBR loading</li>
<ol type="A">
<ol type="A">
<li>Suspend 10 uL (100 ug) beads in 100 uL wash buffer </li>
<li>Suspend 10 uL (100 ug) beads in 100 uL Dynabuffer</li>
<li>Apply magnet for 30 sec and remove supernatant</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>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 5 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>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>
</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 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>Mix 20 uL aqueous into 200 uL oil mixture</li>
<li>Vortex for 30 seconds</li>
<li>Vortex for 30 seconds</li>

Revision as of 20:24, 12 April 2017

Emulsion Test-Variables (Started Yesterday)

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Bead Loading Test

Recommended info on Dynabeads

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 Dynabuffer
    2. Apply magnet for 30 sec and remove supernatant
    3. Suspend 10 uL (100 ug) beads in 20 uL Dynabuffer (conc. 5 ug/uL)
    4. Add 20 uL 10 uM template oligo per sample to bead solution; incubate at RT for 5 min
    5. Wash beads with 100 uL wash buffer; vortex to suspend; apply magnet and remove supernatant
    6. Add 100 uL of Wash buffer with 1 uL of 100:1 diluted SYBR gold (Final dilution 1:10000)
    7. Wash beads with 100 uL wash buffer; vortex to suspend; apply magnet and remove supernatant
    8. Repeat wash step 2X more
    9. Resuspend beads in 100 uL TE buffer
  3. Oil/aqueous mixing (adopted from Shao/Church-adjusted for Church protocol bead concentration)
    1. Mix 20 uL aqueous into 200 uL oil mixture
    2. Vortex for 30 seconds
    3. Repeat mixing to 100 uL (5 times)
    4. Mix with vortexer for 3 minutes
    5. Dilute with pure mineral oil for imaging (1:100)