Daniel:Protocols/MagBeads: Difference between revisions

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(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

Back to Main

Protocol for use with NEB magnetic beads (S1421S). Adapted from the protocol that comes with the beads.

Buffers

  1. Wash Buffer
    1. 0.5 M NaCl
    2. 20 mM Tris-HCl (pH 7.5)
    3. 1 mM EDTA
  2. Elution Buffer
    1. 10 mM Tris-HCl (pH 7.5)
    2. 1 mM EDTA
  3. Low Salt Buffer
    1. 0.15 M NaCl
    2. 20 mM Tris-HCl (pH 7.5)
    3. 1 mM EDTA

Protocol

  1. Biotin-labeled substrate binding
    1. Prewarm Elution Buffer at 70C
    2. Prep a 65C water bath
    3. Place low salt buffer on ice
    4. Dissolve biotin labeled substrate in wash buffer to ~ 8pmol/uL
    5. Aliquot Streptavidin beads to 5:1 (w/w) ratio with substrate
    6. Resuspend beads by adding equal volume wash buffer; vortex to resuspend
    7. Apply magnet to side of the tube for 30 seconds; remove supernatant
    8. Add 25 uL biotin substrate solution to beads; vortex to resuspend
    9. Inucbate at room temperature for 5 minutes
    10. Apply magnet and remove supernatant
    11. Wash beads with 100 uL wash buffer; vortex to suspend; apply magnet and remove supernatant
  2. Target binding
    1. Dissolve target in 50 uL wash buffer and heat at 65C for 5 minutes
    2. Chill on ice for 3 minutes
    3. Add sample to prepared strep beads; vortex to suspend
    4. Incubate at RT for 10 minutes with occasional agitation by hand
    5. Apply magnet and remove supernatant
    6. Add 100 uL wash buffer and vortex to suspend beads; apply magnet and remove supernatant
    7. Repeat wash step above
    8. Add 100 uL cold Low Salt Buffer and vortex to suspend; apply magnet and remove supernatant
  3. Elution
    1. Add 25 uL prewarmed elution buffer; vortex to suspend
    2. Incubate at RT for 2 minutes
    3. Apply magnet and transfer supernatant to a clean centrifuge tube
    4. Repeat elution with 25 uL fresh elution buffer; add supernatant to first elution