Plasmids pVAX/fimH(mam) and pVAX/fimH(wt) were constructed in our laboratory as described previously [14 (link)]. Briefly, fimH(mam) comprised of mammalian (mam) codon usage sequences of FimH protein and fimH(wt) composed of wild type (wt) codon usage sequences of FimH protein. The fimH(mam) and fimH(wt) were cloned into the vector pVAX1 containing an anti-kanamycin gene (Invitrogen, Carlsbad, CA, USA) to create pVAX/fimH(mam) and pVAX/fimH(wt) constructs. DNA vaccine was prepared using an Endo Free Plasmid Giga-prep Kit (Qiagen, Hilden, Germany).
Endo free plasmid giga prep kit
Manufactured by Qiagen
Sourced in United States
The Endo Free Plasmid Giga-prep Kit is a laboratory product designed for the large-scale purification of plasmid DNA. It utilizes a silica-based membrane technology to efficiently isolate high-quality, endotoxin-free plasmid DNA from bacterial cultures.
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3 protocols using endo free plasmid giga prep kit
1
Construction of Mammalian Codon FimH Plasmids
2
Interleukin-2 Plasmid Electrotransfection in Mice
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For the electrotransfection experiments, pUNO1-mIL02 (InvivoGen, Toulouse, France) plasmid coding for mouse interleukin 2 was used. The plasmid was propagated in E. coli DH5α and purified using the EndoFree Plasmid GigaPrep kit (Qiagen, Chatsworth, CA, USA) according to the manufacturer’s instructions. The extracted plasmid DNA was validated by electrophoresis on agarose gel. The concentration and purity were determined by using Nanodrop 2000 (Thermo Fisher, Washington, DC, USA).
Electrotransfection was done on both tibialis anterior muscles of each treated mouse. Firstly, the back legs of the mice were shaved, and remaining hair was removed with depilation cream. Afterward, 35 µL of the IL-2 coding plasmid solution (diluted in saline solution) at a concentration of 1 mg/mL was injected into the tibialis anterior muscles of sedated mice. Electric fields on the muscle were applied 10 min after the injection. One or both tibialis anterior muscles were electro-transferred during the treatment.
Electrotransfection was done on both tibialis anterior muscles of each treated mouse. Firstly, the back legs of the mice were shaved, and remaining hair was removed with depilation cream. Afterward, 35 µL of the IL-2 coding plasmid solution (diluted in saline solution) at a concentration of 1 mg/mL was injected into the tibialis anterior muscles of sedated mice. Electric fields on the muscle were applied 10 min after the injection. One or both tibialis anterior muscles were electro-transferred during the treatment.
3
Plasmid-based PTH1-34 Gene Expression
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The human PTH1-34 gene (prepro-PTH 1-34) 19 was inserted into a plasmid (pUMVC1) with a CMV promotor to generate pUMVC1/hPTH pDNA. pUMVC1/LacZ was used as a negative control for cAMP ELISA. Endotoxin-free plasmids were prepared using EndoFree plasmid Giga-prep kit (Qiagen, CA, USA).
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