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 (5 pmol/uL; order 10<sup>12</sup> molecules/uL)</li> | |||
<li>Each mg binds ~400 pmol peptide-biotin (say BSA)</li> | |||
<li>Each mg contains ~10<sup>10</sup> beads/mL, which is 10<sup>7</sup> beads/uL</li> | |||
<li>Each uL beads binds | |||
</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=== | |||
<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== | |||
===Buffers=== | |||
<ol> | <ol> | ||
Line 26: | Line 105: | ||
</ol></ol> | </ol></ol> | ||
==Protocol== | ===Practical Buffer Mixes=== | ||
'''Wash/Binding Buffer''' | |||
{| 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="65" height="30" | Reagent | |||
| width="65" | Stock | |||
| width="65" | Final | |||
| width="65" | Dilution | |||
| width="65" | Amt in 10 mL | |||
|- style="font-size:12pt" | |||
|style="font-weight:bold" height="15" valign="bottom" | NaCl | |||
| align="center" valign="bottom" | 1.5 M | |||
| align="center" valign="bottom" | 0.5 M | |||
| align="center" align="center" valign="bottom" | 3 | |||
| align="center" valign="bottom" | 3.33 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" | 20 mM | |||
| align="center" align="center" valign="bottom" | 25 | |||
| align="center" valign="bottom" | 400 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" | 6.25 mL | |||
|} | |||
'''Low Salt Buffer''' | |||
{| 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="65" height="30" | Reagent | |||
| width="65" | Stock | |||
| width="65" | Final | |||
| width="65" | Dilution | |||
| width="65" | Amt in 10 mL | |||
|- style="font-size:12pt" | |||
|style="font-weight:bold" height="15" valign="bottom" | NaCl | |||
| align="center" valign="bottom" | 1.5 M | |||
| align="center" valign="bottom" | 0.15 M | |||
| align="center" align="center" valign="bottom" | 10 | |||
| align="center" align="center" valign="bottom" | 1 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" | 20 mM | |||
| align="center" align="center" valign="bottom" | 25 | |||
| align="center" align="center" valign="bottom" | 400 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" 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" align="center" valign="bottom" | 8.58 mL | |||
|} | |||
===Protocol=== | |||
<ol> | <ol> |
Latest revision as of 19:54, 5 July 2017
Magnetic Beads Purification[edit]
Protocol for use with NEB magnetic beads (S1421S). Adapted from the protocol that comes with the beads.
DynaBeads (ThermoFisher)[edit]
- Supplied at 10 mg/mL
- Each mg binds ~500 pmol ssDNA-biotin conjugate (5 pmol/uL; order 1012 molecules/uL)
- Each mg binds ~400 pmol peptide-biotin (say BSA)
- Each mg contains ~1010 beads/mL, which is 107 beads/uL
- 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]
- 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[edit]
Buffers[edit]
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 |