The OGAB blocks were amplified by PCR using the primers listed in Supplemental Table S6 . PCR was performed using KOD DNA polymerase (Toyobo). An A-protrusion at the 3’ end was added to the obtained PCR fragment using A-attachment Mix (Toyobo) according to the instruction manual. The obtained DNA was ligated into pMD19 (simple) (Takara) using Mighty Mix ligation mixture (Takara), and then was used to transform each of the E. coli strains TOP10, JM109, and DH5α. Transformants of these plasmids were cultivated with LB medium supplemented with 100 μg/ml of carbenicillin (Wako, Japan). OGAB plasmid was purified from 1 ml of the culture using QIAprep Miniprep Kit (Qiagen) according to the instruction manual. The obtained crude plasmid solution was further purified enzymatically using Plasmid Safe DNase (Epicenter) as follows. Crude plasmid (5 μg/50 μl) was added to 6 μl 10 × buffer for Plasmid Safe DNase, 2.4 μl of 25 mM ATP, and 2 μl Plasmid Safe DNase. This reaction mixture was incubated at 37 °C for 1 h, then incubated at 70 °C for 30 min to inactivate the enzymes. The resulting solution was cleaned up by using a conventional column-based cleanup kit and then eluted in a small volume of TE (pH 8.0) (<25 μl).
Pmd19 simple
Manufactured by Takara Bio
Sourced in Japan, United States, China
The PMD19 is a compact and versatile laboratory device designed for DNA and RNA purification. It utilizes a magnetic bead-based technology to efficiently extract and purify targeted genetic materials from a variety of sample types. The PMD19 offers a streamlined and consistent purification process, making it a reliable tool for researchers and scientists in various fields of study.
Automatically generated - may contain errors
Lab products found in correlation
Qiaprep miniprep kit, by Qiagen (1 mentions)
Kod dna polymerase, by Toyobo (1 mentions)
Plasmid safe dnase, by Illumina (1 mentions)
Carbenicillin, by Fujifilm (1 mentions)
Hiscribe t7 quick high yield rna synthesis kit, by New England Biolabs (1 mentions)
Rnaiso plus, by Takara Bio (1 mentions)
Kod plus neo enzyme, by Toyobo (1 mentions)
Dna extraction kit, by Omega Bio-Tek (1 mentions)
One step bacterial active protein extraction kit, by Sangon (1 mentions)
5 protocols using pmd19 simple
1
Plasmid Preparation and Purification
2
High-yield T7 RNA Synthesis of R20 and R266
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R20 and R266 RNA were produced using the HiScribe T7 Quick High Yield RNA Synthesis Kit (New England Biolabs, Ipswich, MA, USA) following the manufacturer’s instructions. The template for R20 was produced by oligo annealing. Sense and antisense oligos with T7 promoter were obtained from Integrated DNA Technologies, Inc. Coralville, Iowa, USA, annealed in a thermocycler, and used directly in T7 transcription. For R266, the template sequence was amplified from viral cDNA by PCR and subcloned into pMD19-simple (Takara Bio, Inc., Kusatsu, Shiga Prefecture, Japan). Purified and linearized plasmids were then used in T7 transcription. The resulting ssRNA products were treated with DNase I, purified using the RNAiso Plus (Takara Bio, Inc., Kusatsu, Shiga Prefecture, Japan) reagent and precipitated with isopropanol. All RNAs were dissolved in a standard 10 mM Tris–1 mM EDTA buffer.
3
Genomic DNA Extraction and TORC2 Promoter Cloning
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The genomic DNA was isolated from the blood samples of Qinchuan cattle using the phenol chloroform method (Sambrook and Russell, 2001) and DNA extraction kit (Omega Bio-tek, Norcross, GA, USA). The extracted DNA was diluted to make the final concentration as 50 ng/μL and stored at −20 °C. Gene specific primers (TORC2-Fragment 1, Table S1 ) were used to amplify a 1990 bp promoter region, including TSS (transcription start site) of the bovine TORC2 gene (NCBI accession No NC_037330.1 from 16477661 to 16487214). Qinchuan cattle genomic DNA was used as a template for the PCR amplification of TORC2 gene promoter, using KOD plus neo enzyme (Toyobo, Tokyo, Japan). Thermocycling (PCR) was performed using three-step cycle conditions with pre-denaturation at 94.0 °C for 5 min followed by 34 cycles of denaturation at 97 °C for 30 s, annealing (Tm of the primers used, see Table S1 ) for 30 s and final extension at 72.0 °C for 45 s. The PCR product was cloned into pMD19 (simple) (Takara, USA).
4
Purification and Characterization of LfsT Protein
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We first purified the LfsT protein. Plasmid pET32a(+) was used to generate a recombinant plasmid, pOEX, for the expression of the lfsT gene in E. coli BL21(DE3).
A total of 1.5 mM isopropyl β-d -1-thiogalactopyranoside (IPTG) was added to induce the overexpression of lfsT. We used a one-step bacterial active protein extraction kit (Sangon Biotech) and a Ni-nitrilotriacetic acid (NTA)-Sefinose column (Bio Basic, Canada) to extract the His-tagged LfsT protein according to the manufacturers’ protocols. A desalting gravity column (Sephadex) was used to purify the target protein.
For electrophoretic mobility shift assays (EMSAs), we amplified the predicted promoter sequences (Table S2 and Data Set S4) of the tested genes with the primers indicated inTable 2 . The PCR fragments (150 to 250 bp) were first cloned into pMD19 (Simple; TaKaRa), and the ligated fragments were then further amplified using the primer pair M13-47 and RV-M. We used an EMSA kit (Molecular Probes) to perform the analysis according to the manufacturer’s instructions. The DNA-protein complexes were separated by 5% nondenaturing polyacrylamide gel electrophoresis. The gel was visualized with an automatic digital gel image analysis system (Tanon 1600R).
A total of 1.5 mM isopropyl β-
For electrophoretic mobility shift assays (EMSAs), we amplified the predicted promoter sequences (Table S2 and Data Set S4) of the tested genes with the primers indicated in
5
Construction of Suicide Conjugative Vectors
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Three suicide conjugative vectors were used in this study: pUAmT14 was constructed previously (Citation19), and pMSST4 and pSNT3 ( Figure 1 ) were constructed as follows: Plasmid pMSST4 was constructed by inserting the spectinomycin resistance cassette into the SspI-DraI site of pMD19-Simple (TaKaRa Biotechnology, Dalian, China). The resistance cassette was obtained from pIJ778 (Citation18) by restriction digestion with SacI and BstBI. This 1281-bp fragment containing aadA (spectinomycin resistance gene) and oriT (origin of transfer) was blunted before ligation. Plasmid pSNT3 was generated by replacing the EcoRI-HindIII fragment of supercos-1 (Stratagene, La Jolla, CA) with an oriT-fragment that was PCR amplified from pIJ773. The primers used in this reaction were oriF (5′-caagaattcGTGTATCCAACGGCGTCAGC-3′; the EcoRI restriction site is underlined) and oriR (5′-aacaagcttTCGAAGTTCCCGCCAGCC-3′; the HindIII restriction site is underlined). oriT: conjugative transfer origin; aadA: spectinomycin resistance gene; bla: carbenicillin resistance gene; neo: kanamycin resistance gene.
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