Exprep plasmid sv mini

Manufactured by GeneAll

The Exprep Plasmid SV mini is a laboratory equipment designed for the extraction and purification of plasmid DNA. It is a compact and efficient tool for researchers and scientists working with recombinant DNA technology.

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Lab products found in correlation

Ligafast rapid dna ligation system, by Promega (2 mentions) Expin gel sv, by GeneAll (2 mentions) Dneasy blood tissue kit, by Qiagen (2 mentions) Top simple dyemix aliquot hot premix, by Enzynomics (1 mentions) Nanovue, by GE Healthcare (1 mentions) Synergy h1, by Agilent Technologies (1 mentions) Psicheck 2 vector, by Promega (1 mentions)

4 protocols using exprep plasmid sv mini

Generating V2 Deletion Plasmids

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To create the V2 deletion plasmids, the pGL4.11 vector cloned with the V2 original construct was amplified by PCR using 5′-phosphorylated primers; S, 5′-GGC ACA CAG AGA TAC GCG CA-3′, and AS, 5′-TGT GTG CGT GTA GGG GGT TA-3′ (Supplementary Fig. 4). PCR was performed with 12.5 µl of SmartGene 2 × pfu Mixed Taq Advanced (SJ Bioscience, Republic of Korea), 9.5 µl of nuclease-free water, 1 µl of primers (10 pmol/µl), and 1 µl of pGL4.11 vector cloned with V2 original construct as template DNA. The PCR conditions were as follows: initialization at 94 °C for 3 min, 22 thermal cycles of 94 °C for 40 s, primer-specific annealing at 61 °C for 30 s, 72 °C for 6 min, and a final elongation step at 72 °C for 5 min.
All PCR products were separated on a 1.5% agarose gel and purified with Expin Gel SV (GeneAll, Republic of Korea). The purified PCR products were cloned into a pGL4.11-T vector (Promega, USA) and ligated by LigaFast Rapid DNA Ligation System (Promega, USA). Plasmid isolation was performed with the Exprep Plasmid SV mini (GeneAll, Republic of Korea). The vector into which the PCR products were inserted was verified by colony PCR.

Lee D.H., Bae W.H., Ha H., Kim W.R., Park E.G., Lee Y.J., Kim J.M., Shin H.J, & Kim H.S. (2024). The human PTGR1 gene expression is controlled by TE-derived Z-DNA forming sequence cooperating with miR-6867-5p. Scientific Reports, 14, 4723.

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Optimized PTGR1 Promoter Analysis

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Genomic DNA (gDNA) was extracted from HepG2 using DNeasy Blood & Tissue Kit (Qiagen, Germany), according to the manufacturer’s instructions. DNA samples were quantitated at 500 ng/µl using the ND-1000 UV–Vis spectrophotometer (NanoDrop, USA). This gDNA was used for PCR amplification with the 2 × TOP simple DyeMix (aliquot)-HOT premix (Enzynomics, Republic of Korea). Each of the three constructs, including the promoter region of PTGR1, was generated by PCR amplification using one sense (S) primer and three anti-sense (AS) primers: S primer (5′-CTG AGA CCA CCT CTC CTT GC-3′), V1 deletion_AS1 primer (5′-GTG TGC GTG TAG GGG GTT AG-3′), V1 original_AS2 primer (5′-CGC GTA TCT CTG TGT GCC TA-3′), and V2 original_AS3 primer (5′-CTT ACA GGA GCC CGA AGG TT-3′) (Supplementary Fig. 4). The PCR conditions were as follows: initialization at 95 °C for 5 min, 40 thermal cycles of 94 °C for 40 s, primer-specific annealing at 60.5 °C for 40 s, 72 °C for 1 min, and a final elongation step at 72 °C for 5 min. All PCR products were purified with the Exprep Plasmid SV mini (GeneAll, Republic of Korea), Sanger sequencing was conducted by Macrogen (Seoul, Republic of Korea) using the four forward primers and four reverse primers listed above.

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Isolation and Purification of Phage DNA

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Host E. coli cells were centrifuged for 1 h at 7370 rcf and 4°C. The supernatant was taken and incubated for 1 h after adding 0.2 volumes of 30% PEG8000 and 3 M NaCl. After incubation, the mixture was re-centrifuged for 1 h at 7370 rcf and 4°C. The precipitated phage was suspended in 1 ml of TE buffer. Phage quantification was determined from the absorbance at 269 nm and 320 nm, and the absorbance of the samples was measured using a microplate reader (Synergy H1, BioTek, Winooski, VT).
The phages were lysed by adding 2 volumes of lysis buffer (1% SDS and 0.2 M NaOH) and incubating the sample in a 70°C water bath for 1 h. After cooling to 25°C, a 1.5 volume of neutralization buffer (3 M sodium acetate) was added and incubated on ice for 1 h, followed by centrifugation for 1 h at 16 000 rcf and 4°C. The supernatant was transferred to a new tube, and two volumes of NT1 binding buffer (Macherey-Nagel, Düren, Germany) were added. The mixture was loaded onto a spin column (Exprep plasmid SV mini, Geneall Biotechnology, Seoul, Korea) and washed with NT3 buffer (Macherey-Nagel). Finally, an appropriate amount of TE buffer (10 mM Tris, 1 mM EDTA) was used to elute the DNA. ssDNA concentration was calculated from the A260 values measured using a nano spectrometer (Nanovue, GE Healthcare, Chicago, IL, USA) according to the following formula:

Lee B.Y., Lee J., Ahn D.J., Lee S, & Oh M.K. (2021). Optimizing protein V untranslated region sequence in M13 phage for increased production of single-stranded DNA for origami. Nucleic Acids Research, 49(11), 6596-6603.

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Genomic DNA Extraction and PCR Amplification

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The DNeasy Blood & Tissue Kit (Qiagen, Germany) was used to extract genomic DNA (gDNA) from human cell line HEK293A. gDNA was used for PCR amplification with 2 × TOP simple DyeMix (aliquot)-HOT premix (Enzynomics, Republic of Korea). The PCR mixture contained with 10 µL of contained with 2 × TOPsimple DyeMix, 7 µL of distilled water, 1 µL of gDNA template, and 1 µL of each primers (10 pmol/µL). The condition of PCR were as follow: an initialization at 95 °C for 5 min, 35 thermal cycles of 94 °C for 40 s, primer-specific annealing temperatures for 40 s, 72 °C for 40 s, and a final elongation step at 72 °C for 5 min. The products of PCR were separated on a 1.5% agarose gel and purified with Expin Gel SV (GeneAll, Republic of Korea). The purified PCR products were cloned into a psi-CHECK2 vector (Promega, USA) by LigaFast Rapid DNA Ligation System (Promega, USA). The Exprep Plasmid SV, mini (GeneAll, Republic of Korea) was used for plasmid isolation.

Lee H.E., Park S.J., Huh J.W., Imai H, & Kim H.S. (2021). The enhancer activity of long interspersed nuclear element derived microRNA 625 induced by NF-κB. Scientific Reports, 11, 3139.

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