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''' | '''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 | <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 | <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 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)
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 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 5 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)