The second-stage PCR products were separated on a 1.5% agarose gel and then purified using GF-1 PCR Clean-up Kit; Vivantis, Shah Alam, Selangor Darul Ehsan, Malaysia. The inserts (∼800-bp-long PCR products) were ligated into the PGEM-T EASY Vector (Promega, Madison, Wisconsin, United States) using T4 DNA ligase (Fermentas, Waltham, Massachusetts, United States). This complex was transformed into DH5α strain of E. coli. The positive colonies (20–30 clones) were checked for the presence of inserts through the approaches of colony PCR and digestion with EcoRI. The plasmid DNA was extracted from the insert-positive colonies using the GF-1 Plasmid DNA Extraction Kit (Vivantis) and commercially sequenced with a Sanger platform by using the M13F and M13R vector primers at Macrogen Company.
Gf 1 plasmid dna extraction kit
Manufactured by Vivantis Technologies
Sourced in Malaysia
The GF-1 Plasmid DNA Extraction Kit is a laboratory equipment designed for the purification of plasmid DNA from bacterial cultures. It utilizes a silica-based membrane technology to efficiently bind and elute plasmid DNA, providing a simple and reliable method for DNA extraction.
Automatically generated - may contain errors
Lab products found in correlation
Pgem t easy vector, by Promega (2 mentions)
Lipofectamine 2000, by Thermo Fisher Scientific (2 mentions)
Gf 1 pcr clean up kit, by Vivantis Technologies (1 mentions)
T4 dna ligase, by Thermo Fisher Scientific (1 mentions)
Gf 1 ambiclean kit, by Vivantis Technologies (1 mentions)
Instaclone pcr cloning kit, by Thermo Fisher Scientific (1 mentions)
Chromas lite 2, by Technelysium (1 mentions)
Mir p081, by Biosettia (1 mentions)
Rapamycin, by Merck Group (1 mentions)
Coomassie brilliant blue r 250, by Thermo Fisher Scientific (1 mentions)
Kanamycin, by Merck Group (1 mentions)
Fluorescein isothiocyanate (fitc), by Merck Group (1 mentions)
Clonejet pcr cloning kit, by Thermo Fisher Scientific (1 mentions)
Ni nta agarose, by Qiagen (1 mentions)
Isopropyl β d 1 thiogalactopyranoside (iptg), by Sinaclon (1 mentions)
T100 thermal cycler, by Bio-Rad (1 mentions)
Nebexpress cell free e coli protein synthesis system, by New England Biolabs (1 mentions)
Kta pure protein purification system, by Cytiva (1 mentions)
Hiscribe t7 quick high yield rna synthesis kit, by New England Biolabs (1 mentions)
Histrap hp, by Cytiva (1 mentions)
13 protocols using gf 1 plasmid dna extraction kit
1
Cloning and Sequencing of PCR Products
2
Cloning and Sequencing of Amplicon
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The specific amplicon was purified with the GF-1 AmbiClean Kit (Vivantis, Malaysia). The products were subsequently cloned into the pGEM-T Easy vector (Promega, USA) according to the manufacturer′s instructions. The recombinant plasmids were transformed into E. coli (DH5α) competent cells and screened for positive colonies. Plasmids were extracted using the GF-1 Plasmid DNA Extraction Kit (Vivantis, Malaysia). All sequencing reactions were performed on both strands using the sequencing service from Macrogen Inc., Seoul, Korea.
3
Phagemid-based scFv Expression Screening
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Phagemid extraction of positive scFv clones was
submitted for the polymerase chain reaction using a GF-1 plasmid DNA
extraction kit (Vivantis; Malaysia) and used as a DNA template. The
forward and reverse primers were LMB3 (5′- CAG GAA ACA GCT
ATG AC-3′) and pHEN seq (5′- CTA TGC GGC CCC ATT CA-3′),
respectively. Expression screening was carried out through western
blot analysis using a hexahistidine tag antibody.
submitted for the polymerase chain reaction using a GF-1 plasmid DNA
extraction kit (Vivantis; Malaysia) and used as a DNA template. The
forward and reverse primers were LMB3 (5′- CAG GAA ACA GCT
ATG AC-3′) and pHEN seq (5′- CTA TGC GGC CCC ATT CA-3′),
respectively. Expression screening was carried out through western
blot analysis using a hexahistidine tag antibody.
4
Cloning Ct-Specific Gene Fragments
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To clone the amplicons of CtPAL and CtCHS into pTZ57R/T vector, InsTAclone™ PCR Cloning Kit (Thermo SCIENTIFIC, # K1213) and competent cells of Escherichia coli strain JM107 were recruited. In brief, based upon blue/white screening, recombinant colonies were selected for DNA extraction by GF-1 Plasmid DNA Extraction Kit (Vivantis). Sequences of isolated region of CtPAL and CtCHS genes were obtained using M13 universal primers (Faza Pajooh Biotech). Sequences were certified by means of Chromas Lite 2.01 (Technelysium) after clipping the vector sequence.
5
Lentiviral Vector Cloning and Validation
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The pLV-miRNA vector, which carried the hsa-mir106b lentivirus and comprised green
fluorescent protein (GFP) in infected E. coli BL21, was utilized to
generate iPS-like cells (mir-p081, Biosettia, San Diego, CA, USA). The E.
coli BL21 colony was cultured in 5 ml of Lysogeny broth (LB) medium
(Sigma-Aldrich, USA) for 16 hours in a 37°C shaker incubator at 180 rpm. To determine the
presence of vector in the bacteria, it was cultured for 24 hours in LB agar medium
containing 100 µg/ml ampicillin. Vector purification from E. coli BL21
colonies was deployed by GF-1 Plasmid DNA Extraction Kit (Vivantis, Malaysia)
instructions.
The quantitative reverse transcriptase polymerase chain
reaction (qRT-PCR) technique was applied to ensure
the accuracy of the extracted pLV-miRNA vector. The
16s-RNA as a bacterial reference gene and mir-106b
primers are listed in Table 1. The sequences of forward
and reverse primers were designed by using GeneRunner
software. The cDNA was synthesized according to the
manufacturer’s instructions (Fermentas, USA). PCR
products were electrophoresed on 1% agarose gel along
with 1 kb DNA Ladder Marker and examined.
fluorescent protein (GFP) in infected E. coli BL21, was utilized to
generate iPS-like cells (mir-p081, Biosettia, San Diego, CA, USA). The E.
coli BL21 colony was cultured in 5 ml of Lysogeny broth (LB) medium
(Sigma-Aldrich, USA) for 16 hours in a 37°C shaker incubator at 180 rpm. To determine the
presence of vector in the bacteria, it was cultured for 24 hours in LB agar medium
containing 100 µg/ml ampicillin. Vector purification from E. coli BL21
colonies was deployed by GF-1 Plasmid DNA Extraction Kit (Vivantis, Malaysia)
instructions.
The quantitative reverse transcriptase polymerase chain
reaction (qRT-PCR) technique was applied to ensure
the accuracy of the extracted pLV-miRNA vector. The
16s-RNA as a bacterial reference gene and mir-106b
primers are listed in Table 1. The sequences of forward
and reverse primers were designed by using GeneRunner
software. The cDNA was synthesized according to the
manufacturer’s instructions (Fermentas, USA). PCR
products were electrophoresed on 1% agarose gel along
with 1 kb DNA Ladder Marker and examined.
6
Overexpression of EBF1 in KKU-213A Cells
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The EBF1 expression vector (EBF1 vector) and control vector (empty vector) were synthesized (Dharmacon Inc., Lafayette, CO, USA) and extracted from Escherichia coli (E. coli) cells containing a EBF1 vector (pLOC-TurboRFP containing EBF1 gene) or a control vector (pLOC-TurboRFP without EBF1 gene). Plasmid DNA extraction from E. coli was performed using GF-1 Plasmid DNA Extraction Kit (Vivantis Technologies, Selangor, Malaysia) following the manufacturer's instructions. KKU-213A cell line was maintained in complete media at 37 °C in a humidified incubator with 5% CO2. Cells were seeded in six-well plates (1.5 × 105 cells/well) and 70-80% confluent cells were transiently transfected with 1 µg of control vector or EBF1 vector using Lipofectamine 2000 (Invitrogen, MA, USA) according to the manufacturer's recommendations. Twenty-four hours after transfection, cells were collected for various experiments. Cells transfected with control vector served as control cells.
7
Overexpression of FoxA1 in Cells
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The FoxA1 expression vector (FoxA1 vector) and control vector (empty vector) were extracted from Escherichia coli cells containing a FoxA1 vector (pLOC-TurboRFP containing FoxA1 gene, Dharmacon Inc., Lafayette, CO, USA) or control vector (empty vector or pLOC-TurboRFP without interested element, Dharmacon Inc., Lafayette, CO, USA), respectively. The plasmid extraction was performed using GF-1 Plasmid DNA Extraction Kit (Vivantis Technologies, Selangor, Malaysia) following the manufacturer’s protocols. Cells were transiently transfected with plasmids using Lipofectamine 2000 (Invitrogen, Carlsbad, CA, USA). After 24 h of transfection, cells were harvested for RNA extraction, immunocytochemistry, cell proliferation, cell invasion and spheroid formation assays.
8
Recombinant Protein Purification Workflow
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The material in this study included GF-1 plasmid DNA extraction kit (Vivantis CO., Malaysia), Clone JET PCR Cloning Kit (Thermo Fisher Scientific, Inc., USA), pET28a, expression vector (Novagen, USA), IPTG (Sinaclon, Iran), kanamycin (Sigma, Germany), Coomassie Brilliant Blue R-250 (Thermo Fisher Scientific, USA), Ni–NTA agarose (Qiagen, USA), Anti-His antibody (Bioligand, USA), IgG-horseradish peroxidase (HPR)-conjugated secondary antibody (Abcam, USA), EDC (G-Biosciences, USA), SANH (Bioworld, USA), FITC (Sigma-Aldrich, USA), 3-MA (Sigma, Germany), and rapamycin (Sigma, Germany).
9
Plasmid DNA Extraction and Visualization
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The isolation of plasmid DNA from the selected bacterial strains was performed by GF-1 plasmid DNA extraction kit (Vivantis, Malaysia). E. coli V517 was used as a positive control. Electrophoresis of the extracted plasmids was performed in a 1% agarose gel and the plasmids were visualized with UV trans-illumination in a Gel Doc apparatus.
10
Genomic Profiling of Carbapenem-Resistant Enterobacteriaceae
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The isolated strains were cultured in LB broth for 18 h at 37°C and then collected for bacterial plasmid extraction, which was performed using the GF-1 Plasmid DNA Extraction Kit (Vivantis, Selangor DE, Malaysia) according to the manufacturer’s instructions. Then, the plasmid DNA extracts were quantitated using a spectrophotometer. The plasmid DNA template was applied to the PCR system to examine three beta-lactamase genes (blaSHV, blaTEM, and blaOXA) [16 (link)] and three carbapenemase genes (blaNDM-1, blaKPC-2, and blaIMP) [17 (link)], all of which are commonly found in CRE. The PCR was performed on a T100 Thermal Cycler (Bio-Rad, Hercules, CA, USA), and the products were visualized using 1% agarose gel electrophoresis.
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