Daniel:Protocols/MagBeads

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Magnetic Beads Purification[edit]

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

DynaBeads (ThermoFisher)[edit]

  1. Supplied at 10 mg/mL
  2. Each mg binds ~500 pmol ssDNA-biotin conjugate (5 pmol/uL; order 1012 molecules/uL)
  3. Each mg binds ~400 pmol peptide-biotin (say BSA)
  4. Each mg contains ~1010 beads/mL, which is 107 beads/uL
  5. Each uL beads binds

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[edit]

  1. Template Binding
    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 15 min
    5. Wash beads with 100 uL wash buffer; vortex to suspend; apply magnet and remove supernatant
  2. Original Protocol-Binding
    1. Resuspend beads in 2x B&W Buffer to a final concentration of 5 μg/μl (twice original volume)
    2. 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
    3. 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
    4. Separate the biotinylated DNA/RNA coated beads with a magnet for 2-3 mins
    5. Wash 2–3 times with a 1x B&W Buffer
    6. 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
  3. Original Protocol-Release/Elution
    1. The biotin-streptavidin bond is broken by harsh conditions
    2. 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
    3. Alternatively, boi the sample for 5 mins in 0.1% SDS for protein dissociation

    NEB[edit]

    Buffers[edit]

    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

    Practical Buffer Mixes[edit]

    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

    Protocol[edit]

    1. Biotin-labeled substrate binding
      1. Prewarm Elution Buffer at 70C (1.5 mL heat block)
      2. Prep a 65C water bath (0.2 mL thermocycler is fine)
      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