Daniel:Protocols/MagBeads: Difference between revisions

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Protocol for use with [https://www.neb.com/products/s1421-hydrophilic-streptavidin-magnetic-beads#tabselect2 NEB magnetic beads] (S1421S). Adapted from the protocol that comes with the beads.
Protocol for use with [https://www.neb.com/products/s1421-hydrophilic-streptavidin-magnetic-beads#tabselect2 NEB magnetic beads] (S1421S). Adapted from the protocol that comes with the beads.


==Buffers==
==DynaBeads ([https://www.thermofisher.com/us/en/home/references/protocols/proteins-expression-isolation-and-analysis/protein-isolation-protocol/dynabeads-myone-streptavidin-c1.html ThermoFisher])==
 
<ol>
<li>Supplied at 10 mg/mL</li>
<li>Each mg binds ~500 pmol ssDNA-biotin conjugate</li>
</ol>
 
'''Dynabeads Buffer Prep'''
 
{| class="wikitable" <hiddentext>generated with [[:de:Wikipedia:Helferlein/VBA-Macro for EXCEL tableconversion]] V1.8</hiddentext>
|- style="background-color:#C4D79B;font-size:12pt;font-weight:bold" align="center"
| width="105" height="30" | Reagent
| width="85" | Stock
| width="85" | Final
| width="85" | Dilution
| width="85" | Amt in 10 mL
 
|- style="font-size:12pt"
|style="font-weight:bold" height="15"  valign="bottom" | NaCl
| align="center" valign="bottom" | 5 M
| align="center" valign="bottom" | 2 M
| align="center" align="center" valign="bottom" | 2.5
| align="center" valign="bottom" | 4 mL
 
|- style="background-color:#D9D9D9;font-size:12pt"
|style="font-weight:bold" height="15"  valign="bottom" | Tris-HCl
| align="center" valign="bottom" | 500 mM
| align="center" valign="bottom" | 10 mM
| align="center" align="center" valign="bottom" | 50
| align="center" valign="bottom" | 200 uL
 
|- style="font-size:12pt"
|style="font-weight:bold" height="15"  valign="bottom" | EDTA
| align="center" valign="bottom" | 0.5M
| align="center" valign="bottom" | 1 mM
| align="center" align="center" valign="bottom" | 500
| align="center" valign="bottom" | 20 uL
 
|- style="background-color:#D9D9D9;font-size:12pt"
|style="font-weight:bold" height="15"  valign="bottom" | nf H2O
| align="center" valign="bottom" | NA
| align="center" valign="bottom" | NA
| align="center" valign="bottom" | NA
| align="center" valign="bottom" | 5.8 mL
 
|}
 
===Protocol===
 
 
 
==NEB==
 
===Buffers===


<ol>
<ol>
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|}
|}


==Protocol==
===Protocol===


<ol>
<ol>

Revision as of 17:38, 24 April 2017

Magnetic Beads Purification

Back to Main

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

DynaBeads (ThermoFisher)

  1. Supplied at 10 mg/mL
  2. 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

NEB

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

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

Protocol

  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