The purified PCR product was ligated into the pMD19-T vector (Invitrogen, USA). DH5α cells (Biontex, Munich, Germany) were transformed with the ligation product. Plasmid DNA extracted from single colonies was digested with BamHI and EcoRI. The digested DNA was separated on a 1% agarose gel and clones showing DNA fragments of the expected size were sent to Invitrogen (Shanghai, China) for sequencing. Sequencing results were analyzed using Sequencher (Gene Codes Corporation, Ann Arbor, MI, USA) and BLAST to confirm that the DNA sequence encoding BMP-2 was correctly inserted into pMD19-T. The pMD19-T plasmid bearing the BMP-2 insert was designated as TS-BMP-2.
Pmd19 t vector
Manufactured by Thermo Fisher Scientific
Sourced in United States, China
The PMD19-T vector is a plasmid designed for cloning and expressing genes in mammalian cells. It contains a strong promoter, multiple cloning sites, and a selectable marker for identifying and selecting transfected cells. The core function of the PMD19-T vector is to facilitate the introduction and expression of foreign genes in mammalian cell lines.
Automatically generated - may contain errors
Lab products found in correlation
Sequencher, by Gene Codes (1 mentions)
Goldview, by Takara Bio (1 mentions)
Bacterial genome extraction kit, by Sangon (1 mentions)
E coli bl21 de3, by Takara Bio (1 mentions)
Pet 22b vector, by Thermo Fisher Scientific (1 mentions)
Dna marker, by Takara Bio (1 mentions)
Protein marker, by Takara Bio (1 mentions)
Primestar hs dna polymerase, by Takara Bio (1 mentions)
T4 dna ligase, by Takara Bio (1 mentions)
Quickprep micro mrna purification kit, by GE Healthcare (1 mentions)
Transscript synthesis supermix, by Transgene (1 mentions)
Trizol reagent, by Thermo Fisher Scientific (1 mentions)
4 protocols using pmd19 t vector
1
Cloning and Validation of BMP-2 Construct
2
Cloning and Characterization of GmHDZ20
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The coding sequence (CDS) of GmHDZ20 was downloaded from Phytozome6 and amplified from the cotyledon cDNA of the cco mutant with specific primers (Supplementary Table 1 ) using the following cycling profile: 98°C for 2 min followed by 35 cycles of 98°C for 10 s, 55°C for 30 s, and 68°C for 1 min. The amplified product was further gel purified, cloned into the pMD19-T vector, and sequenced (Invitrogen, Shanghai, China).
The isoelectric point and protein molecular weight were predicted with the ProtParam tool7 . Protein sequence alignment of GmHDZ20 and eight other plant HD-Zip proteins was performed by ClustalW in MEGA version 6.0 with default parameters and viewed with GeneDOC. A Neighbor-joining (NJ) phylogenetic tree was built using MEGA 6.0 with 1000 bootstrap replications. Promoter cis-acting elements of GmHDZ20 were evaluated with the Plant CARE database8 .
The isoelectric point and protein molecular weight were predicted with the ProtParam tool
3
Cloning and Expression of Xanthomonas oryzae Protein
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Xanthomonas oryzae pv. Oryzae (CCTCC AB 91123) was obtained from China-Center for Type Culture Collection, Wuhan University (Wuhan, China), and cultured as previously described (Byul et al. 2011 (link)). Both E. coli DH5α used for gene cloning and E. coli BL21 (DE3) serving as an expression host were purchased from TaKaRa Biotechnology Co. Ltd (Dalian, China). E. coli strains were routinely grown in LB broth or on LB agar plates with 100 µg/ml of ampicillin at 37 °C. The pMD19-T vector and pET-22b (+) vector were purchased from Invitrogen Corp. (Shanghai, China), and used for cloning and expression studies, respectively.
DNA Marker, Protein Marker, GoldView, T4 DNA ligase, PrimeSTAR HS DNA Polymerase, QuickCutTM Nde I and QuickCutTM Xho I restriction enzymes were purchased from TaKaRa Biotechnology Co., Ltd (Dalian, China). A bacterial genome extraction kit, a DNA fragment purification kit, an agarose gel DNA purification kit and a MiniBEST Plasmid Purification Kit were purchased from Sangon Biotechnology Co., Ltd. (Shanghai, China). All aqueous solutions and buffers were prepared with water purified with an in-house Milli-Q Plus System (Millipore, Inc., Billerica, MA, USA). All the other chemicals were of analytical grade.
DNA Marker, Protein Marker, GoldView, T4 DNA ligase, PrimeSTAR HS DNA Polymerase, QuickCutTM Nde I and QuickCutTM Xho I restriction enzymes were purchased from TaKaRa Biotechnology Co., Ltd (Dalian, China). A bacterial genome extraction kit, a DNA fragment purification kit, an agarose gel DNA purification kit and a MiniBEST Plasmid Purification Kit were purchased from Sangon Biotechnology Co., Ltd. (Shanghai, China). All aqueous solutions and buffers were prepared with water purified with an in-house Milli-Q Plus System (Millipore, Inc., Billerica, MA, USA). All the other chemicals were of analytical grade.
4
Cloning and Sequencing of Pc-cathepsin D-like cDNA
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For the full-length Pc-cathepsin D-like cDNA cloning, the total RNA was extracted from the hepatopancreas of P. clarkii using the Trizol reagent (Invitrogen, Carlsbad, CA, USA) and then purified using a QuickPrep Micro mRNA Purification Kit (GE Healthcare, USA). The firststrand cDNA was prepared using the TransScript Synthesis SuperMix (TransGen, Beijing, China) according to the manufacturer's instructions. The nucleotide sequence of the Pccathepsin D-like gene was obtained from the P. clarkii genome library, which was previously constructed in our laboratory (Dai et al., 2017; Zhou et al., 2017) . For cloning the Pc-cathepsin D-like gene, gene-specific primers were designed using partial sequences in the P. clarkii database mentioned above using Oligo 7.0 software (Table 1). Thermal cycling for polymerase chain reaction (PCR) was as followed: 94 °C for 3 min, 35 cycles at 94 °C for 30 s, 55 °C for 35 s and 72 °C for 45 s. The PCR products were subjected to agarose gel (1%) electrophoresis, and the gene of interest was purified for further analysis. The purified fragment was then inserted into the pMD19-T Vector and sequenced by Invitrogen. D r a f t
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