Daniel:Protocols/MagBeads: Difference between revisions
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>Djacobse (Created page with "=Magnetic Beads Purification= Back to Main Protocol for use with [https://www.neb.com/products/s1421-hydrophilic-streptavidin-magnetic-beads#tabselect2 N...") |
>Djacobse |
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<li>Dissolve target in 50 uL wash buffer and heat at 65C for 5 minutes</li> | <li>Dissolve target in 50 uL wash buffer and heat at 65C for 5 minutes</li> | ||
<li>Chill on ice for 3 minutes</li> | <li>Chill on ice for 3 minutes</li> | ||
<li>Add sample to prepared strep beads; vortex to suspend<li> | <li>Add sample to prepared strep beads; vortex to suspend</li> | ||
<li>Incubate at RT for 10 minutes with occasional agitation by hand</li> | <li>Incubate at RT for 10 minutes with occasional agitation by hand</li> | ||
<li>Apply magnet and remove supernatant</li> | <li>Apply magnet and remove supernatant</li> |
Revision as of 15:52, 18 August 2016
Magnetic Beads Purification
Protocol for use with NEB magnetic beads (S1421S). Adapted from the protocol that comes with the beads.
Buffers
- 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
Protocol
- Biotin-labeled substrate binding
- Prewarm Elution Buffer at 70C
- Prep a 65C water bath
- 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