Daniel:Protocols/MagBeads: Difference between revisions
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===Protocol=== | ===Protocol=== | ||
<ol> | |||
<li>Template Binding</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> | |||
</ol> | |||
<li>Original Protocol-Binding</li> | |||
<ol type="A"> | |||
<li>Resuspend beads in 2x B&W Buffer to a final concentration of 5 μg/μl (twice original volume)</li> | |||
<li>To immobilize, add an equal volume of the biotinylated DNA/RNA in H2O to dilute the NaCl concentration in the 2x B&W Buffer from 2M to 1 M for optimal binding</li> | |||
<li>Incubate for 15 mins at room temperature using gentle rotation. Incubation time depends on the nucleic acid length: short oligonucleotides (< 30 bases) require max. 10 mins. DNA fragments up to 1 kb require 15 mins</li> | |||
<li>Separate the biotinylated DNA/RNA coated beads with a magnet for 2-3 mins</li> | |||
<li>Wash 2–3 times with a 1x B&W Buffer</li> | |||
<li>Resuspend to the desired concentration. Binding is now complete. Resuspend the beads with the immobilized DNA/RNA fragment in a buffer with low salt concentration, suitable for downstream applications</li> | |||
</ol> | |||
<li>Original Protocol-Release/Elution</li> | |||
<ol type="A"> | |||
<li>''The biotin-streptavidin bond is broken by harsh conditions''</li> | |||
<li>5 mins incubation at 65°C or 2 mins at 90°C in 10 mM EDTA pH 8.2 with 95% formamide will typically dissociate >96% of immobilized biotinylated DNA</li> | |||
<li>Alternatively, boi the sample for 5 mins in 0.1% SDS for protein dissociation</li> | |||
</ol> | |||
==NEB== | ==NEB== |
Revision as of 17:48, 24 April 2017
Magnetic Beads Purification
Protocol for use with NEB magnetic beads (S1421S). Adapted from the protocol that comes with the beads.
DynaBeads (ThermoFisher)
- Supplied at 10 mg/mL
- Each mg binds ~500 pmol ssDNA-biotin conjugate
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 |
Protocol
- Template Binding
- 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
- Original Protocol-Binding
- Resuspend beads in 2x B&W Buffer to a final concentration of 5 μg/μl (twice original volume)
- To immobilize, add an equal volume of the biotinylated DNA/RNA in H2O to dilute the NaCl concentration in the 2x B&W Buffer from 2M to 1 M for optimal binding
- Incubate for 15 mins at room temperature using gentle rotation. Incubation time depends on the nucleic acid length: short oligonucleotides (< 30 bases) require max. 10 mins. DNA fragments up to 1 kb require 15 mins
- Separate the biotinylated DNA/RNA coated beads with a magnet for 2-3 mins
- Wash 2–3 times with a 1x B&W Buffer
- Resuspend to the desired concentration. Binding is now complete. Resuspend the beads with the immobilized DNA/RNA fragment in a buffer with low salt concentration, suitable for downstream applications
- Original Protocol-Release/Elution
- The biotin-streptavidin bond is broken by harsh conditions
- 5 mins incubation at 65°C or 2 mins at 90°C in 10 mM EDTA pH 8.2 with 95% formamide will typically dissociate >96% of immobilized biotinylated DNA
- Alternatively, boi the sample for 5 mins in 0.1% SDS for protein dissociation
- Wash Buffer
- 0.5 M NaCl
- 20 mM Tris-HCl (pH 7.5)
- 1 mM EDTA
- Elution Buffer
- 10 mM Tris-HCl (pH 7.5)
- 1 mM EDTA
- Low Salt Buffer
- 0.15 M NaCl
- 20 mM Tris-HCl (pH 7.5)
- 1 mM EDTA
- Biotin-labeled substrate binding
- Prewarm Elution Buffer at 70C (1.5 mL heat block)
- Prep a 65C water bath (0.2 mL thermocycler is fine)
- Place low salt buffer on ice
- Dissolve biotin labeled substrate in wash buffer to ~ 8pmol/uL
- Aliquot Streptavidin beads to 5:1 (w/w) ratio with substrate
- Resuspend beads by adding equal volume wash buffer; vortex to resuspend
- Apply magnet to side of the tube for 30 seconds; remove supernatant
- Add 25 uL biotin substrate solution to beads; vortex to resuspend
- Inucbate at room temperature for 5 minutes
- Apply magnet and remove supernatant
- Wash beads with 100 uL wash buffer; vortex to suspend; apply magnet and remove supernatant
- Target binding
- Dissolve target in 50 uL wash buffer and heat at 65C for 5 minutes
- Chill on ice for 3 minutes
- Add sample to prepared strep beads; vortex to suspend
- Incubate at RT for 10 minutes with occasional agitation by hand
- Apply magnet and remove supernatant
- Add 100 uL wash buffer and vortex to suspend beads; apply magnet and remove supernatant
- Repeat wash step above
- Add 100 uL cold Low Salt Buffer and vortex to suspend; apply magnet and remove supernatant
- Elution
- Add 25 uL prewarmed elution buffer; vortex to suspend
- Incubate at RT for 2 minutes
- Apply magnet and transfer supernatant to a clean centrifuge tube
- Repeat elution with 25 uL fresh elution buffer; add supernatant to first elution
NEB
Buffers
Practical Buffer Mixes
Wash/Binding Buffer
Reagent | Stock | Final | Dilution | Amt in 10 mL |
NaCl | 1.5 M | 0.5 M | 3 | 3.33 mL |
Tris-HCl | 500 mM | 20 mM | 25 | 400 uL |
EDTA | 0.5M | 1 mM | 500 | 20 uL |
nf H2O | NA | NA | NA | 6.25 mL |
Low Salt Buffer
Reagent | Stock | Final | Dilution | Amt in 10 mL |
NaCl | 1.5 M | 0.15 M | 10 | 1 mL |
Tris-HCl | 500 mM | 20 mM | 25 | 400 uL |
EDTA | 0.5M | 1 mM | 500 | 20 uL |
nf H2O | NA | NA | NA | 8.58 mL |