AECIIs from patients with sepsis-induced ARDS were harvested and subjected to Chip-PCR using EZ CHIP Kit (Millipore, MI, USA) in accordance with the manufacturer’s protocol. The primers for RT-PCR were: 5’-CCTTTGGTGGATAGGGAGGAGG-3’(forward) and 5’- GGATAAAAGACCTAGCATTTTATCC-3’ (reverse). The experiment obtained a product of 58 bases carrying the predicted TFBS of NR2F2, which was cloned into a simple T-vector (Takara) for sequencing. NR2F2 primary antibody (2 μg; Abcam, Cambridge, UK) was used for immunoprecipitation of the target protein.
T simple vector
Manufactured by Takara Bio
Sourced in China
The T-simple vector is a plasmid designed for cloning and expression of target genes in various host organisms. It features a simple and versatile backbone suitable for a wide range of molecular biology applications.
Automatically generated - may contain errors
Lab products found in correlation
Ez chip kit, by Merck Group (2 mentions)
Taq dna polymerase, by Takara Bio (1 mentions)
T4 dna ligase, by Takara Bio (1 mentions)
Fast pfu dna polymerase, by Takara Bio (1 mentions)
Ta cloning, by Takara Bio (1 mentions)
Trizol, by Merck Group (1 mentions)
Superscript 2 reverse transcriptase, by Promega (1 mentions)
6 protocols using t simple vector
1
Identifying NR2F2 Binding Sites in ARDS
2
Mapping KLF4 Transcription Factor Binding Sites
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HBL52 cells infected with Lv-KLF4 for 72 h were harvested and subjected to ChIP-PCR using the EZ ChIP Kit (Millipore) according to the manufacturer’s instructions. The 52 bp PCR product carrying the predicted KLF4 TFBS was amplified and subjected to gelelectrophoresis and cloned into a simple T-vector (Takara Bio) for sequencing. The primers used for RT-PCR are provided in Supplementary Table S1 . KLF4 primary antibody (4 μg; Abcam, UK) was used for purification of the KLF4 protein.
3
Cloning and Expression of VmHEP Genes
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Targeted genes were amplified from the cDNA library using Fast‐Pfu DNA polymerase (Takara, Dalian, China). PCR products (VmHEP genes or signal peptide sequences) were added to an A tail with Taq DNA Polymerase (Takara, Dalian, China) and cloned into a T‐simple vector by means of TA cloning (Takara, Dalian, China). Constructed plasmids were sequenced for confirmation by Sangon Biotech, Xi'an, China. The correct T‐VmHEP genes were digested with corresponding restriction enzymes (ClaI and SalI), and the correct T‐signal peptide sequences were digested with corresponding restriction enzymes (EcoRI and XhoI) at 37 °C for 4 h. These VmHEP genes were finally cloned into the PGR106 (PVX) vector (ClaI and SalI), and the signal peptide sequences were finally cloned into the pSUC2 vector (EcoRI and XhoI) with T4 DNA ligase at 22 °C for 3 h (Takara, Dalian, China) (Giraldo and Valent, 2013 ). The final constructed plasmids were sequenced for confirmation by Sangon Biotech, Xi'an, China.
4
Cloning and Characterization of TIFY Genes
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Total RNA was extracted from G. arboreum seedling tissues using Trizol (Sigma-Aldrich) according to the manufacturer’s instructions. Samples were reverse transcribed using an oligo (dT) primer and SuperScript II reverse transcriptase (Promega, USA). The resulting product was used as a PCR template to amplify the predicted TIFY open reading frames using PrimeSTAR DNA Polymerase. The TIFY cDNA regions were cloned into a T-simple vector (TaKaRa). The accuracy of all clones was confirmed by sequencing. TIFY-specific primers were designed, synthesized, and used to clone the genes. For plant transformations, the TIFY cDNA samples were inserted into the modified pSuper1300 plant transformation vector (provided by Zhen Su from China Agricultural University) under the control of the cauliflower mosaic virus 35S promoter.
5
Isolation and Cloning of CRYBB1 and CRYAA
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The total cDNA of the human lens was obtained using the standard methods as described elsewhere [17 (link)]. The coding sequences of CRYBB1 and CRYAA were isolated from the human lens cDNA with PCR using the following primers: WT CRYBB1 primers (F: 5ʹ-GGA ATT CCA TAT GAT GTC TCA GGC TGC AAA GGC CT-3ʹ, R: 5ʹ-CGC GGA TCC TCA CTT GGG GGG CTC TGT GG-3ʹ) and CRYAA primers (F: 5ʹ-AAT CCA TGG ACA TCG CCA TCC ACC-3ʹ, R: 5ʹ-TAC CTC GAG TTT CTT GGG GGC TGC-3ʹ). The sense and anti-sense primers for the construction of the MU CRYBB1 were 5ʹ-GGA ATT CCA TAT GAT TCT CAG GCT GCA AAG GCC T-3ʹ and 5ʹ-CGC GGA TCC CTA CAT CTG TGG CTG GAA GGC TCC-3ʹ, respectively. The PCR product was cloned in a T-simple vector (Takara, Beijing, China) and sequenced. For protein expression, the coding sequences of CRYBB1 and CRYAA were then inserted between the NdeI/BamHI sites and the NcoI&XhoI sites, respectively, in pET28a vectors for the expression constructs.
6
Rapid 3' RACE for PLK1
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After first-strand cDNA synthesis, TdT (Terminal transferase) (Life Technologies) and dCTP (Life Technologies) was used to append a poly C + -tail to the cDNA. Anchor primer UAP poly G and PLK1 GSP1 were used for the first around amplification of dC-tailed cDNA.
Nested PCR was performed with 2 μl of the first reaction using the primer UAP and PLK1 GSP2. PCR products were examined by gel electrophoresis, and then cloned into T simple vector (Takara) for sequencing.
Nested PCR was performed with 2 μl of the first reaction using the primer UAP and PLK1 GSP2. PCR products were examined by gel electrophoresis, and then cloned into T simple vector (Takara) for sequencing.
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