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=Magnetic Beads Purification= [[Daniel Jacobsen|Back to Main]] 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. ==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> <li>Wash Buffer</li> <ol type="i"> <li>0.5 M NaCl</li> <li> 20 mM Tris-HCl (pH 7.5)</li> <li> 1 mM EDTA</li> </ol> <li>Elution Buffer</li> <ol type="i"> <li> 10 mM Tris-HCl (pH 7.5)</li> <li> 1 mM EDTA</li> </ol> <li>Low Salt Buffer</li> <ol type="i"> <li>0.15 M NaCl</li> <li> 20 mM Tris-HCl (pH 7.5)</li> <li> 1 mM EDTA</li> </ol></ol> ===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> <li>Biotin-labeled substrate binding</li> <ol type="A"> <li>Prewarm Elution Buffer at 70C (1.5 mL heat block)</li> <li>Prep a 65C water bath (0.2 mL thermocycler is fine)</li> <li>Place low salt buffer on ice</li> <li>Dissolve biotin labeled substrate in wash buffer to ~ 8pmol/uL</li> <li>Aliquot Streptavidin beads to 5:1 (w/w) ratio with substrate</li> <li>Resuspend beads by adding equal volume wash buffer; vortex to resuspend</li> <li>Apply magnet to side of the tube for 30 seconds; remove supernatant</li> <li>Add 25 uL biotin substrate solution to beads; vortex to resuspend</li> <li>Inucbate at room temperature for 5 minutes</li> <li>Apply magnet and remove supernatant</li> <li>Wash beads with 100 uL wash buffer; vortex to suspend; apply magnet and remove supernatant</li> </ol> <li>Target binding</li> <ol type="A"> <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>Add sample to prepared strep beads; vortex to suspend</li> <li>Incubate at RT for 10 minutes with occasional agitation by hand</li> <li>Apply magnet and remove supernatant</li> <li>Add 100 uL wash buffer and vortex to suspend beads; apply magnet and remove supernatant</li> <li>Repeat wash step above</li> <li>Add 100 uL cold Low Salt Buffer and vortex to suspend; apply magnet and remove supernatant</li> </ol> <li>Elution</li> <ol type="A"> <li>Add 25 uL prewarmed elution buffer; vortex to suspend</li> <li>Incubate at RT for 2 minutes</li> <li>Apply magnet and transfer supernatant to a clean centrifuge tube</li> <li>Repeat elution with 25 uL fresh elution buffer; add supernatant to first elution</li> </ol></ol>
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