Human AQP4 cDNA cloned into pDEST47 (Life Technologies) was used as previously described (11 (link)). An untagged AQP4 construct was created from this by mutagenesis of the first two codons of the GFP linker peptide to stop codons. These were used as templates for mutagenesis following the QuikChange protocol (Stratagene). Mutagenic primers were synthesized by Sigma. Mutant plasmids were amplified in TOP10 Escherichia coli with 100 ng/ml of ampicillin selection. Plasmid DNA was purified using a Wizard Maxiprep kit (Promega) and diluted to 1 mg/ml for transfection.
Wizard maxiprep kit
Manufactured by Promega
The Wizard Maxiprep kit is a laboratory equipment product designed for the rapid and efficient purification of high-quality plasmid DNA from bacterial cultures. The kit utilizes a silica-based membrane technology to capture and purify plasmid DNA, providing a reliable method for DNA extraction and preparation.
Automatically generated - may contain errors
3 protocols using wizard maxiprep kit
1
Generating Untagged AQP4 Constructs
2
Plasmid DNA Extraction and Analysis
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Plasmid DNA was isolated by Birnboim–Doly alkaline lysis method (Birnboim and Doly, 1979 (link)). Purified through LMP (low melting point) agarose and agarase AgarACE™ (Promega), plasmid DNA was analysed by 1% agarose gel electrophoresis. The plasmid was digested with the restriction endonucleases Kzo9I, RsaI and AluI (Promega). Later, Kzo9I, RsaI and AluI fragments were cloned into pGEM-3Zf(+) (Promega). The recombinant plasmid DNA was then transformed into Escherichia coli DH5α and isolated with a Wizard MaxiPrep kit (Promega). DNA sequencing was performed using universal M13 primers with a BigDye® Terminator v3.1 Cycle Sequencing Kit (Applied Biosystems) on a 3730 DNA Analyser (Applied Biosystems) according to the manufacturer’s instructions.
3
Constructing insect chitinase-containing plasmid
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The plasmid pChi-SB containing the bar gene as a selectable marker and insect chitinase was constructed Fig. 9 . The bar gene was cut from the plasmid pAB8 (4799 bp) with HindIII and inserted in the HindIII site of pAHC25. The insect chitinase gene was also inserted in the plasmid pAHC25 by using EcoRI. The large scale preparation of constructed pChi-SB has been done by using the Wizard maxi prep kit (Promega) for plasmid isolation. The plasmid DNA was diluted to a final concentration of 1μg/μl and used for plant transformation.
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