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Nucleospin gel

Manufactured by Takara Bio
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The NucleoSpin Gel is a gel extraction and purification kit designed for the efficient extraction and purification of DNA fragments from agarose gels. The kit utilizes a silica-based membrane technology to selectively bind DNA, allowing for the rapid and reliable purification of DNA fragments.

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Lab products found in correlation

Pcr clean up kit, by Takara Bio (11 mentions) Q5 high fidelity 2x master mix, by New England Biolabs (2 mentions) Pcr blunt 2 topo vector, by Thermo Fisher Scientific (2 mentions) Protoscript 2 reverse transcriptase, by New England Biolabs (2 mentions) Abi 3130xl dna sequencer, by Thermo Fisher Scientific (2 mentions) Takara pcr human papillomavirus typing set, by Takara Bio (2 mentions) Epitaq hs, by Takara Bio (2 mentions) Qiaamp dna mini kit, by Qiagen (2 mentions) Pcr thermal cycler dice touch, by Takara Bio (2 mentions) Premix sequence analysis, by Takara Bio (2 mentions) Kod fx neo dna polymerase, by Toyobo (2 mentions) Gflex dna polymerase, by Takara Bio (1 mentions) Gel doc ez imaging system, by Bio-Rad (1 mentions) Mupid 2plus, by Takara Bio (1 mentions) Phenol chloroform isoamyl alcohol, by Nacalai Tesque (1 mentions) T4 dna ligase, by New England Biolabs (1 mentions) Nucleospin plasmid easypure, by Macherey-Nagel (1 mentions) Phsg396, by Takara Bio (1 mentions) Nucleospin gel and pcr clean up, by Macherey-Nagel (1 mentions) Pcr clean up, by Takara Bio (1 mentions)

Spelling variants of this product

NucleoSpin Gel and PCR Clean-up kit (92 citations) NucleoSpin Gel and PCR Clean-up (36 citations) Nucleospin gel purification kit (3 citations) Nucleospin Gel and PCR purification kit (3 citations) NucleoSpin® Gel and PCR Clean-Up columns (2 citations) Nucleospin Gel and PCR kit (2 citations) NucleoSpin Gel & PCR Clean-up kit (2 citations) Nucleospin gel extraction kit (2 citations) Nucleospin® Gel and PCR Clean-up Midi (2 citations)

14 protocols using nucleospin gel

1

Characterization of ORF10 and ORF61 Transcripts

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Total RNA was reverse transcribed using ProtoScript II Reverse
Transcriptase (New England Biolabs, MA, USA). Gene-specific reverse
transcription primers TV_004 and TV_007 were used for ORF10 and ORF61
transcripts, respectively. Traditional PCR was used to confirm the
existence of ORF10 and ORF61 transcripts using the following primers:
ORF10_c3244 forward TV_017, reverse TV_004; ORF10_c2410 forward TV_018,
reverse TV_004; ORF61_c8714 forward TV_025, reverse TV_010; ORF61_c20295
forward TV_026, reverse TV_010. Each PCR reaction mixture contained 12.5
μL Q5® High-Fidelity 2X Master Mix (NEB), 10 μM
each primer, 1 μL cDNA, and nuclease-free water to a final volume
of 25 μl. PCR was performed with pre-denaturation at 98°C
for 30 s, amplification with 35 cycles of denaturation at 98°C
for 10 s, annealing at 56°C for 30 s, and extension at
72°C for 15 s, followed by a final extension at 72°C for 2
min. PCR products were loaded onto 1.5% agarose gels and the expected
bands were excised and purified according to the NucleoSpin Gel and PCR
Clean-Up kit instructions (Clontech/Takara, Kyoto, Japan). The purified
PCR products were cloned into pCR-Blunt II-TOPO Vector
(Invitrogen/Thermofisher). Subsequently, DNA minipreps were prepared
from ten colonies per culture and Sanger sequenced by Genewiz (NJ,
USA).
O’Grady T., Feswick A., Hoffman B.A., Wang Y., Medina E.M., Kara M., van Dyk L.F., Flemington E.K, & Tibbetts S.A. (2019). Genome-wide Transcript Structure Resolution Reveals Abundant Alternate Isoform Usage from Murine Gammaherpesvirus 68. Cell reports, 27(13), 3988-4002.e5.
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2

Characterization of ORF10 and ORF61 Transcripts

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Total RNA was reverse transcribed using ProtoScript II Reverse
Transcriptase (New England Biolabs, MA, USA). Gene-specific reverse
transcription primers TV_004 and TV_007 were used for ORF10 and ORF61
transcripts, respectively. Traditional PCR was used to confirm the
existence of ORF10 and ORF61 transcripts using the following primers:
ORF10_c3244 forward TV_017, reverse TV_004; ORF10_c2410 forward TV_018,
reverse TV_004; ORF61_c8714 forward TV_025, reverse TV_010; ORF61_c20295
forward TV_026, reverse TV_010. Each PCR reaction mixture contained 12.5
μL Q5® High-Fidelity 2X Master Mix (NEB), 10 μM
each primer, 1 μL cDNA, and nuclease-free water to a final volume
of 25 μl. PCR was performed with pre-denaturation at 98°C
for 30 s, amplification with 35 cycles of denaturation at 98°C
for 10 s, annealing at 56°C for 30 s, and extension at
72°C for 15 s, followed by a final extension at 72°C for 2
min. PCR products were loaded onto 1.5% agarose gels and the expected
bands were excised and purified according to the NucleoSpin Gel and PCR
Clean-Up kit instructions (Clontech/Takara, Kyoto, Japan). The purified
PCR products were cloned into pCR-Blunt II-TOPO Vector
(Invitrogen/Thermofisher). Subsequently, DNA minipreps were prepared
from ten colonies per culture and Sanger sequenced by Genewiz (NJ,
USA).
O’Grady T., Feswick A., Hoffman B.A., Wang Y., Medina E.M., Kara M., van Dyk L.F., Flemington E.K, & Tibbetts S.A. (2019). Genome-wide Transcript Structure Resolution Reveals Abundant Alternate Isoform Usage from Murine Gammaherpesvirus 68. Cell reports, 27(13), 3988-4002.e5.
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3

CRISPR-Cas3 DNA Cleavage Assay

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To detect in vitro DNA cleavage activity of CRISPR-Cas3 proteins, targeted sequences of EMX1 with PAM variants (5′-AAG-3′ or 5′-CCA-3′) were cloned into a pCR4Blunt-TOPO plasmid vector (Thermo Fisher Scientific) according to the manufacturer’s protocol. For collateral DNA cleavage assays, 60 bp activator fragments of hEMX1 and mTyr (which included a target site) were designed and purchased. Targeted sequences for CRISPR-Cas3, CRISPR-Cas12a, and CRISPR-Cas9 are listed in Supplementary Data 2. PAM sequence variants and targeted sequence variants were also designed to examine collateral ssDNA cleavage activity. Biotin-labeled fragments were also purchased for protein-DNA interaction analysis. For fragment analysis, fluorescence-labeled primers were designed and the DNA fragment amplified from genomic DNA of HEK293T cells using Gflex DNA polymerase (Takara-bio). Amplicons were purified using NucleoSpin Gel and a PCR Clean-up kit (Takara-bio) according to the manufacturer’s protocols. A DNA fragment for hs-AFM was also amplified with non-labeled primers. All sequences of primers and donor DNAs are listed in Supplementary Table 3 and Supplementary Data 2, respectively.
Yoshimi K., Takeshita K., Kodera N., Shibumura S., Yamauchi Y., Omatsu M., Umeda K., Kunihiro Y., Yamamoto M, & Mashimo T. (2022). Dynamic mechanisms of CRISPR interference by Escherichia coli CRISPR-Cas3. Nature Communications, 13, 4917.
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4

Purification and Analysis of RPA Products

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After an RPA reaction on the QCM plate, 100 µL of the solution in the QCM cell was collected. The solution was then purified using a NucleoSpin Gel and a PCR Clean-up kit (TaKaRa Bio Inc., Shiga, Japan), which allowed the purification of DNA products from RPA solutions by removing proteins using a spin column method (Figure S1). The purified DNA solution was mixed with Novel Juice as a staining reagent and analyzed by electrophoresis on a 3% agarose gel in TBE Buffer with Mupid-2plus (TaKaRa Bio Inc., Shiga, Japan). After electrophoresis, the gel was photographed using a Gel Doc EZ Imaging System (Bio-Rad Laboratories Inc., Hercules, CA, USA).
Kumagai H, & Furusawa H. (2024). Real-Time Monitoring of a Nucleic Acid Amplification Reaction Using a Mass Sensor Based on a Quartz-Crystal Microbalance. Biosensors, 14(4), 155.
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5

HPV Genotyping Using PCR and Sequencing

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HPV genotypes were identified as follows: Genomic DNA (10 ng) was amplified using PCR for two distinct HPV genomic regions. The E6/E7 region of HPV was amplified using the primer set pU-1M/pU2R (HPVpU-1M: 5′-TGTCAAAAACCGTTGTGTCC-3′ and HPVpU-2R: 5′-GAGCTGTCGCTTAATTGCTC-3′); the region containing the HPV L1 gene was amplified using the primer set GP5+/GP6+ (GP5+: 5′-TTTGTTACTGTGGTAGATACTAC-3′, GP6+: 5′-GAAAAATAAACTGTAAATCATATTC-3′). PCR reactions were performed using the TaKaRa PCR Human Papillomavirus Typing Set (TaKaRa Bio Inc., Shiga, Japan). PCR products were purified using the NucleoSpin Gel (TaKaRa Bio Inc.) or a PCR Clean-up Kit (TaKaRa Bio Inc.). Sanger sequencing was performed using an ABI 3130xl DNA Sequencer (Applied Biosystems, Foster City, CA, USA), according to the manufacturer’s instructions. Similarity between the obtained sequences and various HPV genotypes in the GenBank database was determined by Basic Local Alignment Search Tool (BLAST) analysis (https://blast.ncbi.nlm.nih.gov/Blast.cgi (accessed on 17 June 2019)).
Takayanagi D., Hirose S., Kuno I., Asami Y., Murakami N., Matsuda M., Shimada Y., Sunami K., Komatsu M., Hamamoto R., Kato M.K., Matsumoto K., Kohno T., Kato T., Shiraishi K, & Yoshida H. (2021). Comparative Analysis of Genetic Alterations, HPV-Status, and PD-L1 Expression in Neuroendocrine Carcinomas of the Cervix. Cancers, 13(6), 1215.
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6

DNA Extraction from Mucosal Swabs

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An overview of this method is presented in Figure 1C. The swab samples in the SNET buffer, including mucosae, were incubated at 56 °C for 30 min to disassemble the protein, followed by vigorous shaking. Subsequently, the SNET buffer, including mucosae DNA, was eluted from the swab samples by centrifugation at 5000× g for 1 min at room temperature, via the use of a cell strainer. The DNA pellets were obtained from 800 µL of the eluted solutions by applying equal volumes of phenol/chloroform/isoamyl alcohol (Cat# 25970-56; Nacalai Tesque, Nakagyo, Kyoto, Japan) with a general centrifugation and precipitation method. The pellets were then dissolved in 50 µL of Milli-Q water containing Ribonuclease A (10 µg/mL) and incubated at 37 °C for 10 min to degrade residual RNA. The solutions were then purified using NucleoSpin Gel and a PCR clean-up kit (Cat# U0609B; Takara Bio, Kusatsu, Shiga, Japan), the latter of which was used according to the manufacturer’s instructions; the final elution volume was 30 µL. The purified DNA solutions were subjected to gel electrophoresis to confirm the degree of degradation with a positive control of intact DNA that was extracted from adipose-derived mesenchymal stem cells, called the 1117 cell (our original), of a Toy Poodle.
Sugasawa T., Matsumoto Y., Fang H., Takemasa T., Komine R., Tamai S., Gu W., Tanaka K., Kanki Y, & Takahashi Y. (2023). Establishing a Sequencing Method for the Whole Mitochondrial DNA of Domestic Dogs. Animals : an Open Access Journal from MDPI, 13(14), 2332.
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7

Bisulfite Conversion and Sequencing

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Bisulfite modification of the purified DNA was performed using a MethylEasy Xceed Rapid DNA Bisulphite Modification Kit (Human Genetic Signatures, North Ryde NSW, Australia) according to the manufacturer’s instructions. The converted DNA was amplified by polymerase chain reaction (PCR) with TaKaRa EpiTaq HS (Takarabio, Shiga, Japan), Bisulfite PCR Fw, and Bisulfite PCR Rv. The following PCR protocol was used: 98°C for 3 min and 35 cycles each at 98°C for 10 s, 55°C for 15 s, and 72°C for 1 min. The PCR products were purified with NucleoSpin Gel and a PCR Clean-up kit (Takara) and sequenced using Bisulfite sequence Rv. The primers used in this study are listed in Table S2.
Nishimura A., Kumagai T., Nakatani M, & Yoshimura K. (2016). Method for selective quantification of adipose-derived stromal/stem cells in tissue. Journal of Biological Methods, 3(4), e58.
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8

Carbapenemase Gene Cloning and Evaluation

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Cloning was performed by amplifying the suspected carbapenemase gene using a primer set targeting the first and last 20 bases of the gene with added restriction sites XbaI and EcoRI. The resulting fragment was cleaned using the commercial kit Nucleospin gel and PCR cleanup (MACHEREY-NAGEL GmbH & Co, Düren, Germany). The clean fragment was subjected to cutting by XbaI and EcoRI restriction enzyme (New England Biolabs, Ipswich, MA, USA) and cleanup, as stated above. The vector pHSG396 (Takara Bio, Saint-Germain-en-Laye, France) was subjected to the same restriction enzymes and separated by agarose gel electrophoresis, followed by Nucleospin gel and PCR cleanup kit. The vector and insert were ligated by T4 DNA Ligase (New England Biolabs). The circular plasmid with the gene was inserted into MAX Efficiency™ DH10β Competent Cells (Invitrogen, Waltham, MA, USA). The resulting plasmids were extracted using NucleoSpin® Plasmid EasyPure (MACHEREY-NAGEL). The success of cloning was evaluated by positive PCR for the target gene. The modified carbapenem inactivation method was used on the C. freundii isolate, the transformant E. coli DH10β with the suspected carbapenemase ORF, and E. coli DH10β with the pHSG396 plasmid but no insert (as a negative control).
Frenk S., Rakovitsky N., Kon H., Rov R., Abramov S., Lurie-Weinberger M.N., Schwartz D., Pinco E., Lellouche J, & Carmeli Y. (2021). OXA-900, a Novel OXA Sub-Family Carbapenemase Identified in Citrobacter freundii, Evades Detection by Commercial Molecular Diagnostics Tests. Microorganisms, 9(9), 1898.
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9

RNAi Template Amplification and dsRNA Synthesis

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Target genes for RNAi were amplified using cDNA and used as a template using gene-specific primers (Table S4). To amplify templates for target dsRNA, PCR was performed in a total volume of 25 μL using 1 μL of cDNA, 12.5 μL of 2 × Master Mix (Emerald, Takara), and 0.5 μL of 10 μM for gene-specific primers. The reaction was amplified with the following PCR conditions: initial denaturation was carried out for 5 min at 95 °C, then 35 cycles of 45 s at 95 °C, 56 °C, and 72 °C were performed. The process was then extended for 10 min at 72 °C. The amplified products were resolved on 1% agarose gel followed by PCR product or gel cleanup using NucleoSpin® Gel and a PCR Clean-up kit (Takara Bio, USA). The quantification of purified PCR products was performed using a Spectrophotometer (Eppendorf) and products were stored at −20 °C until further processing. The double-stranded RNA (dsRNA) of target genes and green fluorescence protein (GFP, control) was synthesized by using a MEGAscript™ RNAi Kit. The integrity of the dsRNA was analyzed on 1% agarose gels, and the concentration was determined by a Spectrophotometer (Eppendorf, Pocklington, York, UK).
Sandal S., Singh S., Bansal G., Kaur R., Mogilicherla K., Pandher S., Roy A., Kaur G., Rathore P, & Kalia A. (2023). Nanoparticle-Shielded dsRNA Delivery for Enhancing RNAi Efficiency in Cotton Spotted Bollworm Earias vittella (Lepidoptera: Nolidae). International Journal of Molecular Sciences, 24(11), 9161.
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10

Methylation Profiling of Ctse in Mice

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Genomic DNA isolated from CD4+ T cells of MRL (n = 5) and B6 (n = 5) mice was bisulphite converted using the MethylEasy Xceed Rapid DNA Bisulphite Modification Kit (TaKaRa) following the manufacturer’s protocol. Cytosines are converted to uracils whereas 5-methylcytosines are unreactive. The 440 bp fragment within 583 bp region at intron 1 of Ctse was amplified by nested PCR with EpiTaq HS (TaKaRa) and a set of primers: Primer F1: 5′-TAGGTATGTTTTTTATTTATATTTTTAGTG-3′ and Primer R1: 5′-GACTAAATAAATTTTCTTTTTCTAATACTT-3′. PCR products were gel purified with NucleoSpinGel and PCR clean-up (TaKaRa) kits. DNA direct sequencing was performed with BigDye Terminator v3.1 Cycle Sequencing kit (Thermo Fisher Scientific) and methylation patterns at the level of individual CpG sites were examined.
Furthermore, the purified PCR products from CD4+ T cells of MRL (n = 1) and B6 (n = 1) mice were individually cloned into a T-vector pD20 using the DNA Ligation Kit Mighty Mix, and transformed to E.coli HST08 Premium Competent Cells (TaKaRa). The 72 plasmid clones in B6 and 65 in MRL were isolated and sequenced.
Hiramatsu S., Watanabe K.S., Zeggar S., Asano Y., Miyawaki Y., Yamamura Y., Katsuyama E., Katsuyama T., Watanabe H., Takano-Narazaki M., Matsumoto Y., Kawabata T., Sada K.E, & Wada J. (2019). Regulation of Cathepsin E gene expression by the transcription factor Kaiso in MRL/lpr mice derived CD4+ T cells. Scientific Reports, 9, 3054.
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