p3×FLAG-CMV-9 and p3×FLAG-CMV-10 vectors were purchased from MilliporeSigma. The pAP1(1)-Luc vector was purchased from Affymetrix, Inc.; pGL4.75[hRluc CMV] was from Promega Corporation; and pNCMO2 was from Takara Bio, Inc.
Pncmo2
Manufactured by Takara Bio
Sourced in Japan, China
The PNCMO2 is a compact, high-performance laboratory centrifuge from Takara Bio. It is designed for a wide range of centrifugation applications in life science research. The PNCMO2 can accommodate various rotor configurations to accommodate different tube sizes and sample volumes.
Automatically generated - may contain errors
Lab products found in correlation
Brevibacillus choshinensis, by Takara Bio (2 mentions)
Pny326, by Takara Bio (2 mentions)
E coli jm109, by Takara Bio (2 mentions)
Revertaid first strand cdna synthesis kit, by Thermo Fisher Scientific (2 mentions)
Pgl4.75 hrluc cmv, by Promega (1 mentions)
Neomycin, by Merck Group (1 mentions)
Hrp conjugated goat anti mouse igg, by Transgene (1 mentions)
Bovine serum albumin (bsa), by Merck Group (1 mentions)
E coli dh5α cells, by Thermo Fisher Scientific (1 mentions)
E coli dh5α competent cells, by Sangon (1 mentions)
Pmd19 t, by Takara Bio (1 mentions)
Neomycin, by Solarbio (1 mentions)
Plasmid mini kit, by Omega Bio-Tek (1 mentions)
Gel extraction kit, by Omega Bio-Tek (1 mentions)
7 protocols using pncmo2
1
Plasmid Vectors for Cellular Assays
2
Recombinant LOX-1 Protein Expression
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B. choshinensis SP3 (Takara, Japan) was used for protein expression. The B. choshinensis-E. coli shuttle vector pNCMO2 (Takara) was used for expression in B. choshinensis. A site-directed mutagenesis kit was purchased from Stratagene (Shanghai, China). Recombinant human LOX-1 protein was purchased from Sino Biological Inc., China. Anti-His tag antibody and HRP-conjugated goat anti-mouse IgG were purchased from TransGen Biotech (Beijing, China). Bovine serum albumin (BSA) and neomycin were purchased from Sigma-Aldrich, UK. TM medium with modifications (3% glucose, 2.2% polypeptone, 0.8% meat beef extract, 0.2% yeast extract, 0.001% FeSO4·7H2O, 0.001% MnSO4·4H2O, and 0.0001% ZnSO4·7H2O) was used to culture the B. choshinensis strains.
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B. choshinensis SP3 (Takara, Japan) was used for protein expression. The B. choshinensis-E. coli shuttle vector pNCMO2 (Takara) was used for expression in B. choshinensis. A site-directed mutagenesis kit was purchased from Stratagene (Shanghai, China). Recombinant human LOX-1 protein was purchased from Sino Biological Inc., China. Anti-His tag antibody and HRP-conjugated goat anti-mouse IgG were purchased from TransGen Biotech (Beijing, China). Bovine serum albumin (BSA) and neomycin were purchased from Sigma-Aldrich, UK. TM medium with modifications (3% glucose, 2.2% polypeptone, 0.8% meat beef extract, 0.2% yeast extract, 0.001% FeSO4·7H2O, 0.001% MnSO4·4H2O, and 0.0001% ZnSO4·7H2O) was used to culture the B. choshinensis strains.
3
Developing Organic Solvent-Tolerant Bacillus Whole-Cell Cofactor Regenerators
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Bacillus subtilis 168, previously reported to exhibit an organic solvent-tolerant property (Siriphongphaew et al. 2012 (link)), was selected as a host for development of a whole-cell cofactor regenerator. The gdh fragments were amplified from the plasmid pJET-gdh-bs and pJET-gdh-ba using primers containing restriction sites for BsrGI and KpnI (Table 1 ; primers no. 3, 6 and 7). After a double digestion with BsrGI and KpnI, the fragments were inserted into a Bacillus expression vector pHP2N. Plasmid pHP2N was constructed based on a plasmid backbone of E. coli-Bacillus shuttle vector pHY300PLK (TaKaRa, Shiga, Japan) with an insertion of a strong P2 promoter from a commercial expression plasmid pNCMO2 (TaKaRa, Shiga, Japan). The resulting plasmids, named as pHGBS and pHGBA, were introduced into B. subtilis 168 by electroporation using a protocol described previously (Siriphongphaew et al. 2012 (link)). Successful transformation was checked by a colony PCR as well as a restriction analysis. The recombinant B. subtilis 168 overexpressing GluDH-BS and GluDH-BA were referred as B. subtilis BS and B. subtilis BA, respectively.
4
Phospholipase D Expression in Brevibacillus
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Brevibacillus choshinensis (Takara Bio Inc.) was used to express phospholipase D. Plasmid pNY326 and pNCMO2 (Takara Bio Inc.) were used as the expression vector. The strain E. coli JM109 (Takara Bio Inc.) was used as the cloning host. Other chemicals, unless otherwise noted, were purchased from Sinopharm Chemical Reagent Co. Ltd. (SCRC; Shanghai, China).
5
Cultivation and Manipulation of B. choshinensis
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B. choshinensis strain HB116 (Takara Bio, Inc.) was used in this study. E. coli DH5α competent cells (Sangon Biotech Co, Ltd.) were used for DNA manipulation. pNCMO2 (Takara Bio, Inc.) and pMD19-T (Takara Bio, Inc.) were used as the vector and subcloning plasmid, respectively. Milk-Tween (MT) medium containing 2% yeast extract, 10% glucose, 10% polypeptone, 5% meat extract, 0.01 % FeSO4·7H2O, 0.001% ZnSO4·6H2O and 0.01% MnSO4·4H2O was used to culture strain HB116. E. coli DH5α cells were cultured in Luria Broth medium (Oxoid; Thermo Fisher Scientific, Inc.). NaOH was used to adjust the pH of all media to 7.0. Neomycin (20 µg/ml; Beijing Solarbio Science & Technology Co, Ltd.) was added to the media used to culture bacteria containing pNCMO2 and derivatives.
6
Cloning a Full-Length Hepatopancreas Gene
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The scallops were purchased from the Nanshan seafood market in Qingdao. The hepatopancreas was dissected by hand from the scallop viscera. Then, the hepatopancreas was ground into powder in liquid nitrogen. Next, the total RNA was extracted from the powder using the Mollusc RNA Kit (Omega Bio-tek, Norcross, GA, USA). A cDNA library of scallop hepatopancreas was constructed by reverse transcription, using the extracted total RNA as a template (RevertAid First Strand cDNA Synthesis Kit, Thermo Fisher Scientific, Waltham, MA, USA). Using the cDNA library as a template, a full-length gene of Lcf without the signal peptide was cloned using the following primers: a forward primer (5′-CGGGATCCGCAGGCTTCCGTGACGATTTCAC-3′) and a reverse primer (5′-CCGCTCGAGTCAATGAGGTATCATCTCTATGTAATC-3′). Target gene fragments were collected using the Gel Extraction Kit (Omega Bio-tek, Norcross, GA, USA), and ligated into the shuttle vector pNCMO2 (Takara, Dalian, China) using the restriction enzymes BamHI and XhoI. Then, the ligation solution was transformed into E. coli JM109. Expression plasmids were extracted using the Plasmid Mini Kit (Omega Bio-tek, Norcross, GA, USA).
7
Phospholipase D Expression in Brevibacillus
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Brevibacillus choshinensis (Takara Bio Inc.) was used to express phospholipase D. Plasmid pNY326 and pNCMO2 (Takara Bio Inc.) were used as the expression vector. The strain E. coli JM109 (Takara Bio Inc.) was used as the cloning host. Other chemicals, unless otherwise noted, were purchased from Sinopharm Chemical Reagent Co. Ltd. (SCRC; Shanghai, China) Cultivation medium and conditions E. coli JM109 was cultured on the Luria broth (LB) medium containing 10.0 g/L tryptone, 5.0 g/L yeast extract, and 10.0 g/L NaCl, with the introduction of 50 mg/L Ampicillin (Amp) to each culture. In the shake-ask culture, the primary inoculum of B. choshinensis was grown for 24 h in the 250-mL ba ed asks containing 50 mL of TM medium at 30 ℃ and 120 rpm in a shaker until OD 600 = 2~3.
Subsequently, the seed culture (0.5 mL) was diluted to 50 mL of medium A in a 250-mL shake ask at 30 ℃ and 120 rpm. The TM medium contained 10.0 g/L glucose, 10.0 g/L tryptone, 5.0 g/L beef extract, 2.0 g/L yeast extract, 10.0 mg/L FeSO
Subsequently, the seed culture (0.5 mL) was diluted to 50 mL of medium A in a 250-mL shake ask at 30 ℃ and 120 rpm. The TM medium contained 10.0 g/L glucose, 10.0 g/L tryptone, 5.0 g/L beef extract, 2.0 g/L yeast extract, 10.0 mg/L FeSO
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