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Nucaway

Manufactured by Thermo Fisher Scientific
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NucAway is a lab equipment product designed to facilitate the removal of nucleic acids from samples. It functions as a spin column-based system for the purification and concentration of nucleic acids.

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Lab products found in correlation

Mmessage mmachine sp6 kit, by Thermo Fisher Scientific (1 mentions) Megashortscript t7 kit, by Thermo Fisher Scientific (1 mentions) Doxorubicin, by Merck Group (1 mentions)

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NucAway Spin Columns (76 citations) NucAway columns (7 citations)

8 protocols using nucaway

1

Synthesis of Poly-Biotin cAMP

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Example 2

The cAMP-oligo described in Example 1 (0.6 nmole) was mixed with 0.9 nmole of Targext99

(5′-TAT ATT ATA TTA TAT TAT ATT ATA TTA TAT TAT ATT
ATA TTA TAT TAT ATT ATA TTA TAT TAT ATT ATA TTA TAT
TAT ATC TCG ATC CAT GAC CTC AGC-3′).
The mixture contained 43 nmoles biotin-16-dUTP and 76 nmoles each of dATP, dCTP and dGTP in 10 mM Tris-HCl, pH 7.9, 10 mM MgCl2, 1 mM DTT and 50 mM NaCl. T4 DNA polymerase exo minus (13 units) (Lucigen, Middleton, Wis.) was added, and the mixture was incubated at 37° C. for 90 min. The reaction was stopped with the addition of EDTA to a final concentration of 12.5 mM. The resulting poly-biotin cAMP was separated from free nucleotides using a NucAway (Ambion, Foster City, Calif.) size exclusion spin column equilibrated with phosphate buffered saline (PBS).

US11162939B2. Multisignal reagents for labeling analytes (2021-11-02). Enzo Life Science, Inc. [US]. Inventors: Jack Coleman [US], Maciej Szczepanik [US], Richard Jin [US].
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2

RNA-Protein Binding Assay with Radiolabeled Probe

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Single stranded RNA probes were in vitro transcribed using 32P-UTP incorporation with T7 polymerase (MegaScript, Ambion) followed by removal of unincorporated nucleotides (NucAway, Ambion). 10 pmol of labeled probe was mixed with 10 μg tRNA in binding buffer (10% glycerol, 20 mM NaCl, 60 mM KCl, 20 mM HEPES pH 7.5) and heated to 95° for 2 min, then rapidly cooled on ice. 47 pmol protein was added and incubated on ice for 30 min. Samples were run at 4° initially at 1000 V for 1.5 min then at 250 V for approximately 3 h on a pre-run 6% GTG acrylamide gel (90 mM Tris, 30 mM taurine, 0.5 mM EDTA).
Chen D., Brovkina M., Matzat L.H, & Lei E.P. (2019). Shep RNA-Binding Capacity Is Required for Antagonism of gypsy Chromatin Insulator Activity. G3: Genes|Genomes|Genetics, 9(3), 749-754.
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3

Synthesis of Poly-Biotin cAMP

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Example 2

The cAMP-oligo described in Example 1 (0.6 nmole) was mixed with 0.9 nmole of Targext99 (5′-TAT ATT ATA TTA TAT TAT ATT ATA TTA TAT TAT ATT ATA TTA TAT TAT ATT ATA TTA TAT TAT ATT ATA TTA TAT TAT ATC TCG ATC CAT GAO CTC AGC-3′) (SEQ ID NO: 2). The mixture contained 43 nmoles biotin-16-dUTP and 76 nmoles each of dATP, dCTP and dGTP in 10 mM Tris-HCl, pH 7.9, 10 mM MgCl2, 1 mM DTT and 50 mM NaCl. T4 DNA polymerase exo minus (13 units) (Lucigen, Middleton, Wis.) was added, and the mixture was incubated at 37° C. for 90 min. The reaction was stopped with the addition of EDTA to a final concentration of 12.5 mM. The resulting poly-biotin cAMP was separated from free nucleotides using a NucAway (Ambion, Foster City, Calif.) size exclusion spin column equilibrated with phosphate buffered saline (PBS).

US10060910B2. Multisignal reagents for labeling analytes (2018-08-28). None [US], None [US], None [US]. Inventors: Jack Coleman [US], Maciej Szczepanik [US], Richard Jin [US].
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4

Synthesis of Biotinylated cAMP Oligonucleotide

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Example 2

The cAMP-oligo described in Example 1 (0.6 nmole) was mixed with 0.9 nmole of Targext99 (5′-TAT ATT ATA TTA TAT TAT ATT ATA TTA TAT TAT ATT ATA TTA TAT TAT ATT ATA TTA TAT TAT ATT ATA TTA TAT TAT ATC TCG ATC CAT GAC CTC AGC-3′) (SEQ ID NO:2). The mixture contained 43 nmoles biotin-16-dUTP and 76 nmoles each of dATP, dCTP and dGTP in 10 mM Tris-HCl, pH 7.9, 10 mM MgCl2, 1 mM DTT and 50 mM NaCl. T4 DNA polymerase exo minus (13 units) (Lucigen, Middleton, Wis.) was added, and the mixture was incubated at 37° C. for 90 min. The reaction was stopped with the addition of EDTA to a final concentration of 12.5 mM. The resulting poly-biotin cAMP was separated from free nucleotides using a NucAway (Ambion, Foster City, Calif.) size exclusion spin column equilibrated with phosphate buffered saline (PBS).

US10184934B2. Multisignal reagents for labeling analytes (2019-01-22). Enzo Life Science, Inc. [US]. Inventors: Jack Coleman [US], Maciej Szczepanik [US], Richard Jin [US].
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5

CRISPR/Cas9 Mutagenesis of Zebrafish sbds Gene

Cited 2 times
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CRISPR/Cas9 mutagenesis was based on the method of Gagnon et al. (61 (link)). Briefly, the web-based software “CHOPCHOP” (http://chopchop.cbu.uib.no) (62 (link)) was used to design a set of 2 sgRNA molecules (designated gRNA^XcmI and gRNA^BbslI, Supplemental Table 2) to target exon 2 of zebrafish sbds. sgRNA was transcribed in vitro using the pCS2-nCas9 (Addgene 47929) and MEGAshortscript-T7 kit (Ambion, Thermo Fisher Scientific). Cas9 was transcribed in vitro using the SP6 mMESSAGE mMACHINE kit (Ambion, Thermo Fisher Scientific). Both reagents were purified using columns (NucAway, Ambion, Thermo Fisher Scientific). sgRNA and Cas9 transcripts were coinjected into 1-cell–stage embryos. Each embryo was injected with approximately 2 nL of solution containing 12.5–25 ng/μL of sgRNA and 300 ng/μL of Cas9 mRNA in 0.3 M KCl (Supplemental Figure 1B). Genomic DNA was extracted from embryos at 1–2 days after injection for restriction site polymorphism-based genotyping (Supplemental Figure 1C). Primers are detailed in Supplemental Table 2. All obtained alleles were characterized by sequencing PCR products obtained from homozygous embryos.
Oyarbide U., Shah A.N., Amaya-Mejia W., Snyderman M., Kell M.J., Allende D.S., Calo E., Topczewski J, & Corey S.J. (2020). Loss of Sbds in zebrafish leads to neutropenia and pancreas and liver atrophy. JCI Insight, 5(17), e134309.
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6

Biotinylated cAMP Synthesis Protocol

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Example 2

The cAMP-oligo described in Example 1 (0.6 nmole) was mixed with 0.9 nmole of Targext99

(5′-TAT ATT ATA TTA TAT TAT ATT ATA TTA TAT TAT
ATT ATA TTA TAT TAT ATT ATA TTA TAT TAT ATT ATA
TTA TAT TAT ATC TCG ATC CAT GAC CTC AGC-3′).
The mixture contained 43 nmoles biotin-16-dUTP and 76 nmoles each of dATP, dCTP and dGTP in 10 mM Tris-HCl, pH 7.9, 10 mM MgCl2, 1 mM DTT and 50 mM NaCl. T4 DNA polymerase exo minus (13 units) (Lucigen, Middleton, Wis.) was added, and the mixture was incubated at 37° C. for 90 min. The reaction was stopped with the addition of EDTA to a final concentration of 12.5 mM. The resulting poly-biotin cAMP was separated from free nucleotides using a NucAway (Ambion, Foster City, Calif.) size exclusion spin column equilibrated with phosphate buffered saline (PBS).

US11073512B2. Multisignal reagents for labeling analytes (2021-07-27). Enzo Life Sciences, Inc. [US]. Inventors: Jack Coleman [US], Maciej Szczepanik [US], Richard Jin [US].
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7

Roquin-1 Binding to RNA: EMSA Protocol

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RNA fragments for electromobility shift assays (EMSAs) were radioactively labeled using T4 polynucleotide kinase and [γ32P]ATP. Sepharose spin columns (NucAway; Ambion) were used to separate the RNA from free nucleotides. The radioactively labeled RNA (5 nm), the Roquin-1 protein (aa 2–441) and the tRNA competitor (30 μg/mL) were incubated in Hepes/NaCl/MgCl2 (HNM) buffer (10 mM HEPES pH 7.5, 150 mM NaCl, 2 mM MgCl2) and 4% glycerol in the final volume of 20 μL for 30 min at 25 °C. In competition experiments, unlabeled competitor RNA was added afterwards and incubated for another 15 min at 25 °C. The samples were resolved by native Tris/Borate/EDTA (TBE) PAGE (4% polyacrylamide, 1× TBE buffer). Gels were analyzed using radiograph films or Fuji Imaging plates exposed in the FLA-3000, after 15 min incubation in fixing solution (30% (vol/vol) methanol, 10% (vol/vol) acetic acid) and vacuum drying. The signal was quantified using ImageJ (version 1.51). The curve fitting and the calculation of the KDs was done using the software Origin (version 9.0.0G).
Rehage N., Davydova E., Conrad C., Behrens G., Maiser A., Stehklein J.E., Brenner S., Klein J., Jeridi A., Hoffmann A., Lee E., Dianzani U., Willemsen R., Feederle R., Reiche K., Hackermüller J., Leonhardt H., Sharma S., Niessing D, & Heissmeyer V. (2018). Binding of NUFIP2 to Roquin promotes recognition and regulation of ICOS mRNA. Nature Communications, 9, 299.
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8

Doxorubicin Intercalation Kinetics in RNA Nanoparticles

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Doxorubicin (Sigma) solution (20 μM) was incubated with extended Endo28-3WJ-Sph1 or Scramble-3WJ-sph1 nanoparticles (2 μM) in the intercalation buffer (0.1 M sodium acetate, 0.05 M NaCl, 0.01 M MgCl2) for 1 hour at room temperature. The free Doxorubicin was then removed from the system by passing through Sephadex G50 spin column (NucAway™, Ambion). The drug loading efficiency was monitored by measuring the fluorescence intensity of Doxorubicin with a fluorescence spectrophotometer (Horiba Jobin Yvon) at excitation wavelength of 480 nm and emission from 500 to 720 nm.
To measure the intercalation constant of Endo28-3WJ-Sph1 nanoparticle with Doxorubicin, increasing concentrations of RNA nanoparticles were incubated with 1.4 μM Doxorubicin, and the fluorescence intensity of Doxorubicin was measured. The fluorescence quenching was plotted as a function of Dox-conjugating aptamer concentration and fitted to Hill equation in Origin to calculate the Kd.
Pi F., Zhang H., Li H., Thiviyanathan V., Gorenstein D.G., Sood A.K, & Guo P. (2016). RNA Nanoparticles Harboring Annexin A2 Aptamer Can Target Ovarian Cancer for Tumor-Specific Doxorubicin Delivery. Nanomedicine : nanotechnology, biology, and medicine, 13(3), 1183-1193.
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