Human A1AT cDNA (GenBank accession number: NM_000295) was purchased from the Korea Human Gene Bank (Daejeon, South Korea). The hA1AT gene was subcloned into a TA vector (Enzynomics, South Korea) using two restriction sites of Hind III and Xho I. Site‐directed mutagenesis was conducted for the F51S mutation using an EzChange site‐directed mutagenesis kit according to the manufacturer's instructions (Enzynomics). The primers used for polymerase chain reaction were: forward primer, 5′‐caccaatatctCcttctccccagtg‐3′ and reverse primer, 5′‐gagaagGagatattggtgctgttggac‐3′. The mutated nucleotides are shown in capital letters. The sequences of constructs were confirmed by DNA sequencing.
Ta vector
Manufactured by Enzynomics
Sourced in Cameroon
The TA vector is a DNA cloning vector that is commonly used for the insertion and expression of genes in bacterial and eukaryotic systems. It has a single 3' terminal thymidine (T) overhang that facilitates the direct ligation of PCR products, which typically have 3' adenine (A) overhangs. The TA vector is a useful tool for molecular biology research and applications that require the efficient cloning of PCR amplified DNA fragments.
Automatically generated - may contain errors
Lab products found in correlation
Histrap, by GE Healthcare (3 mentions)
Q sepharose, by GE Healthcare (2 mentions)
Ezchange site directed mutagenesis kit, by Enzynomics (1 mentions)
Superdex 200 10 300 gl, by GE Healthcare (1 mentions)
Hiprep 26 10 desalting, by GE Healthcare (1 mentions)
Capto q, by GE Healthcare (1 mentions)
4 4 dianilino 1 1 binaphthyl 5 5 disulfonic acid bis ans, by Merck Group (1 mentions)
Pfu polymerase, by Enzynomics (1 mentions)
Hiprep, by Cytiva (1 mentions)
Capto, by Cytiva (1 mentions)
Hitrap desalting column, by GE Healthcare (1 mentions)
Hitrap, by Cytiva (1 mentions)
Iscript cdna synthesis kit, by Bio-Rad (1 mentions)
Mx3000p qpcr system, by Agilent Technologies (1 mentions)
Sybr premix ex taq reagent, by Takara Bio (1 mentions)
Ex taq polymerase, by Takara Bio (1 mentions)
Taq polymerase, by Enzynomics (1 mentions)
Restriction enzymes, by Enzynomics (1 mentions)
P nitrophenyl pnp esters, by Merck Group (1 mentions)
7 protocols using ta vector
1
Cloning and Mutagenesis of hA1AT Gene
2
Recombinant Sphingomonas sp. Protein Expression
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Sphingomonas sp. PAMC 26621 was provided by the Polar and Alpine Microbial Collection of the Korea Polar Research Institute (Incheon, South Korea) [18 (link)]. The pET28 expression vector was purchased from Novagen (Madison, WI, USA). The TA vector, restriction enzymes, and pfu polymerase were acquired from Enzynomics (Daejeon, South Korea). HisTrap, Capto Q, HiPrep desalting 26/10, Superdex 200 10/300 GL, and Superdex 200 prep grade XK16 columns were purchased from GE Healthcare (Piscataway, NJ, USA). 4,4′-Dianilino-1,1′-binaphthyl-5,5′-disulfonic acid (bis-ANS) was purchased from Sigma (St. Louis, MO, USA). All other chemical reagents were purchased from Sigma or Tokyo Chemical Industry (Tokyo, Japan).
3
Cloning and mutagenesis of Sphingomonas grxC
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The strain Sphingomonas sp. PAMC 26621, isolated from the Svalbard Island in the Arctic Ocean, was kindly provided by the Polar and Alpine Microbial Collection at the Korea Polar Research Institute (Incheon, South Korea) [18 (link)]. The polymerase chain reaction (PCR) was employed to amplify the 258-bp grxC (spgrx3) gene (NCBI ID: NZ_AIDW01000021.1:c56439-56182) from the genome of Sphingomonas sp. PAMC 26621, which was subsequently subcloned into a TA vector (Enzynomics, Daejeon Korea). After digestion with Nde I and EcoR I enzymes TA–spgrx3 construct, the pET28–spgrx3 construct was inserted into a pET28 vector (Novagen, Madison, WI, USA) and then introduced into Escherichia coli BL21 (DE3) through transformation. Site-directed mutagenesis of the pET28–spgrx3 construct was performed using pfu polymerase and PCR primers listed in S1 Table . After PCR amplification, the products were subjected to Dpn I digestion at 37°C for 1 h to remove the template plasmids. The resulting PCR products were then transformed into E. coli BL21 (DE3). The DNA sequences of both wild-type (WT) and mutant plasmids were validated through DNA sequencing analysis.
4
Heterologous Expression of Sphingomonas sp.
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Sphingomonas sp. PAMC 26621 was provided by the Polar and Alpine Microbial Collection (PAMC) of the Korea Polar Research Institute (Incheon, Korea) [14 (link)]. The pET28b (+) expression vector was purchased from Novagen (Madison, WI, USA), the TA vector was from Enzynomics (Daejeon, South Korea), and the HisTrap, Q Sepharose, and HiTrap desalting columns were from GE Healthcare (Piscataway, NJ, USA). All of the other reagents were purchased from Sigma unless stated otherwise.
5
Cloning and Mutagenesis of Sphingomonas grxD Gene
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Sphingomonas sp. PAMC 26621 was kindly provided by the Polar and Alpine Microbial Collection of the Kore Polar Research Institute (Incheon, South Korea) [22 ]. The 333‐bp grxD (spgrx4) gene (NCBI ID: WP_010164075.1) was amplified from the genome of Sphingomonas sp. PAMC 26621 by PCR and subcloned into a TA vector (Enzynomics, Daejeon, South Korea). The TA–spgrx4 construct, digested by Nde I and BamH I, was subcloned into a pET28 vector (Novagen, Madison, WI, USA) and transformed into Escherichia coli BL21 (DE3). The pET28–spgrx4 construct was used as a template for site‐directed mutagenesis by PCR using pfu polymerase (PCR primers listed in TableS1 ). The PCR products were incubated with Dpn I at 37 °C for 1 h to remove the template plasmids before transformation into E. coli BL21 (DE3). The DNA sequence of the WT and mutant plasmids was confirmed by DNA sequencing.
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Sphingomonas sp. PAMC 26621 was kindly provided by the Polar and Alpine Microbial Collection of the Kore Polar Research Institute (Incheon, South Korea) [22 ]. The 333‐bp grxD (spgrx4) gene (NCBI ID: WP_010164075.1) was amplified from the genome of Sphingomonas sp. PAMC 26621 by PCR and subcloned into a TA vector (Enzynomics, Daejeon, South Korea). The TA–spgrx4 construct, digested by Nde I and BamH I, was subcloned into a pET28 vector (Novagen, Madison, WI, USA) and transformed into Escherichia coli BL21 (DE3). The pET28–spgrx4 construct was used as a template for site‐directed mutagenesis by PCR using pfu polymerase (PCR primers listed in Table
6
Characterization of the GAZ Gene
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Approximately 0.5 mg of total RNA was used for cDNA synthesis using the iScript cDNA Synthesis Kit according to the manufacturer's instructions (Bio-Rad, USA). The cDNA samples were then subjected to the qRT-PCR assays using SYBR Premix ExTaq reagent (Takara, Japan) and the Mx3000P QPCR system (Agilent Technologies, USA). The UBQ10 gene was used as the internal reference. RT-PCR was conducted using ExTaq polymerase (Takara, Japan), and the PCR amplification was carried out with 35 cycles for GAZ and 30 cycles for UBQ10 and ACT2. The amplified PCR products were loaded on 1% agarose gel (Intron Biotechnologies, South Korea). Each experiment was independently conducted with at least three biological replicates. The PCR primer sequence information is listed in Supplemental Table 2.
To identify the 5 0 region of the GAZ gene, 5 0 RACE was carried out using a 5 0 /3 0 RACE Kit (Roche, USA) according to the manufacturer's instructions. RNA samples extracted from 7-day-old WT roots were used with a series of nested GAZ-specific primers. The primer sequences are listed in Supplemental Table 3. The final PCR products were cloned into the TA vector (Enzynomics, South Korea) and sequenced with M13 forward and reverse primers.
To identify the 5 0 region of the GAZ gene, 5 0 RACE was carried out using a 5 0 /3 0 RACE Kit (Roche, USA) according to the manufacturer's instructions. RNA samples extracted from 7-day-old WT roots were used with a series of nested GAZ-specific primers. The primer sequences are listed in Supplemental Table 3. The final PCR products were cloned into the TA vector (Enzynomics, South Korea) and sequenced with M13 forward and reverse primers.
7
Characterization of Sphingomonas glacialis
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Sphingomonas glacialis PAMC 26605, isolated from the Arctic lichen Ochrolechia sp., was provided by the Polar and Alpine Microbial Collection (PAMC) of Korea Polar Research Institute (Korea) [23] . The strain, formerly known as Sphingomonas sp. PAMC 26605 [24] , was renamed S. glacialis PAMC 26605 in the EzTaxon database [25] , based on its 16S rRNA sequence homology to S. glacialis C16y T (99.78%) [26] . Taq polymerase, TA vector, and restriction enzymes were purchased from Enzynomics (Korea). pET28a(+) expression vector was acquired from Novagen (USA). HisTrap, Q-Sepharose, and Superdex-200 HR 10/300 gel filtration columns were purchased from GE Healthcare (Piscataway, USA), and p-nitrophenyl (pNP) esters were purchased from Sigma (USA). Aspirin (acetylsalicylic acid), salicylic acid, acetaminophen, and 4-aminophenol were acquired from TCI (Japan). All other reagents were from Sigma unless noted otherwise.
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