The gene for human PDI in the pUC57 vector was purchased from GenScript Biotech Corporation, New Jersey, U.S. Later, it was sub-cloned into the pET-28b (+) (Novagen, U.S.A.) within the Nhe I and Hind III restriction sites, a 6X His-tagged was also placed at the N-terminal. To get the optimum expression in a bacterial expression system, the codon was optimized. The resulting gene was transformed in Escherichia coli DH5α competent cells to amplify the copy number of the plasmid. Colonies were picked, followed by incubation in the primary culture, and then the plasmid was isolated by using a mini-prep plasmid isolation kit (Qiagen, Hilden, Germany). Plasmid was run on 0.8% agarose gel before and after digestion with Nhe I and Hind III restriction enzymes (Supplementary Figure S1A and 1B), which was further confirmed by Sanger's sequencing performed by Macrogen, Seoul, South Korea.
Mini prep plasmid isolation kit
Manufactured by Qiagen
Sourced in Germany
The Mini-prep plasmid isolation kit is a laboratory tool designed to extract and purify plasmid DNA from bacterial cultures. It provides a straightforward and efficient method for obtaining small-scale plasmid samples.
Automatically generated - may contain errors
5 protocols using mini prep plasmid isolation kit
1
Cloning and Characterization of Human PDI
2
Construction of pVAX1-SAG1 Recombinant Plasmid
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The pPrime vector carrying the SAG1 gene and the eukaryotic expression vector pVAX1 (Invitrogen, Carlsbad, USA) were digested by NdeI and XhoI restriction enzymes. After thermal inactivation of the restriction enzymes and analysis by LMP agarose gel electrophoresis, the linearized pVAX1 plasmid and the SAG1 gene were extracted from the %1 agarose gel by agarose gel DNA extraction kit (Qiagen, Hilden, Germany). The purified linear vector and insert were subjected to ligation reaction using T4 DNA ligase (Roche, Mannheim, Germany). E. coli DH5α competent cells were transformed with 2 uL of ligation product. Transformed E. coli cells were selected on LB medium agar plates containing kanamycin (50 mg/L). Several colonies were assayed by colony PCR using specific primers (SAG1F and SAG1R). After selecting recombinant clones, the plasmid DNA was extracted from the cells cultured overnight by using the Miniprep plasmid isolation kit (Qiagen, Hilden, Germany) and confirmed by PCR, restriction-enzyme digestion, followed by DNA sequencing using T7 and BGH primers.
3
Purification and Quantification of pEGFP-C2
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Enhanced plasmid DNA pEGFP-C2 (4.7 kb) was isolated from E. coli DH5α and purified using a Qiagen miniprep
plasmid isolation kit. The concentration of pEGFP was determined using
nanoDrop in a SpectraMax i3x microplate reader. The purity was checked
from the absorbance ratio at 260 and 280 nm.
plasmid isolation kit. The concentration of pEGFP was determined using
nanoDrop in a SpectraMax i3x microplate reader. The purity was checked
from the absorbance ratio at 260 and 280 nm.
4
Overexpression of F11R in OPM-2 Cells
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F11R overexpression was carried out using the pCMV6-hF11R expression vector (Origene, Rockville, MD). Plasmids were isolated using a Qiagen mini-prep plasmid isolation kit (Qiagen Inc., Valencia, CA). OPM-2 cells were transiently transfected with pCMV6-hF11R and the empty vector pCMV6 using the Transfast reagent (Promega, Madison, WI) according to the manufacturer's instructions. Briefly, 1 μg of each plasmid was diluted in serum-free medium. The diluted plasmids were vortexed before the addition of 3 μl Transfast reagent to bring the Transfast/DNA ratio to 1:1. Cells in Transfast/DNA mixture were then incubated at 37°C for 1 h. Positively transfected cells were subsequently selected using puromycin.
5
Generating Cynomolgus Monkey TRIM5α shRNA
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The coding region of the cynomolgus monkey TRIM5α gene was obtained from the NCBI GenBank (https://www.ncbi.nlm.nih.gov/nucleotide) and tested for similarity by performing a BLAST to eliminate potential target sequences to match off-target gene of the same species. The potential single-nucleotide polymorphisms (SNP) were eliminated (http://www.ncbi.nlm.nih.gov/projects/SNP/). Three shRNA targeting different regions of the TRIM5α (shTRIM5α) gene were then selected (Fig. 1. A). The shTRIM5α and nontargeting control shRNA (Table 1) and PCR oligos (Table 2) were synthesized by Dalian Bao Biological Co., Ltd. and puri ed by PAGE.
The lentiviral vector plasmid pLL3.7 was double-digested with EcoRI and BamHI, the linearized vector fragment was recovered, and the double-stranded oligos were annealed transformed into DH5α competent cells, vector extracted using Qiagen miniprep plasmid isolation kit and sequenced at Shanghai Yingke Jieke Company.
The lentiviral vector plasmid pLL3.7 was double-digested with EcoRI and BamHI, the linearized vector fragment was recovered, and the double-stranded oligos were annealed transformed into DH5α competent cells, vector extracted using Qiagen miniprep plasmid isolation kit and sequenced at Shanghai Yingke Jieke Company.
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