For plasmid transfection, a pCMV-lacz plasmid (Plasmid factory) was used encoding a β-galactosidase protein (LacZ) gene under the control of a CMV promoter. The EEV plasmid was created by incorporating a SFV DNA replicase sequence (kindly donated by Prof Greg Atkins, Virus Group, Department of Microbiology, School of Genetics and Microbiology, Trinity College Dublin). A nuclear localization sequence was also incorporated to allow for more efficient nuclear targeting of the pEEV plasmid (Figure 1 ). The pMG plasmid was purchased from InvivoGen (Toulouse, France) (Supplementary Figure S1 ). Plasmids were propagated in Escherichia coli strain Top10 and purified on endotoxin-free Qiagen-tip 500 columns (Qiagen, Manchester, UK).
Tip 500 columns
Manufactured by Qiagen
Sourced in United Kingdom
The Qiagen-tip 500 columns are small, disposable columns designed for the purification of nucleic acids, such as plasmid DNA, genomic DNA, and RNA, from a variety of sample sources. The columns contain a silica-based resin that selectively binds nucleic acids under appropriate buffer conditions, allowing for efficient capture, washing, and elution of the target molecules.
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Lab products found in correlation
3 protocols using tip 500 columns
1
Plasmid Transfection Protocol for Cell Transduction
2
Murine GM-CSF and Human B71 Expression
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The mammalian expression vector pMG was purchased from Invivogen (Cayla SAS, Toulouise, France). A version of this plasmid, designated pGT141, containing the murine GM-CSF and human B71 genes transcriptionally controlled from the hEF1–HTLV and CMV promoters, respectively, was designed and cloning was performed on contract by Invivogen. Human B71 cDNA has been shown previously to function in a murine setting. The inserts were confirmed by sequencing. Plasmids were propagated in Escherichia coli strain Top10 and purified on endotoxin-free Qiagen-tip 500 columns (Qiagen, Manchester, UK).
3
Engineered Plasmid for Cancer Immunotherapy
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The pMG plasmid was purchased from InvivoGen (Toulouse, France). A version of this plasmid, designated pGT141, containing the murine GMCSF and human B7-1 genes transcriptionally controlled from the hEF1-HTLV and cytomegalovirus promoters, respectively, was designed and cloning was performed on contract by InvivoGen. The EEV plasmid was created by incorporating a Semliki Forest virus DNA replicase sequence (kindly donated by Prof Greg Atkins, Virus Group, Department of Microbiology, School of Genetics and Microbiology, Trinity College, Dublin, Ireland). A nuclear localisation sequence was also incorporated to allow for nuclear targeting.26 The murine GMCSF and human b7.1 genes were also incorporated into the pEEV. Plasmids were propagated in Escherichia coli strain Top10 and purified on endotoxin-free Qiagen-tip 500 columns (Qiagen) All plasmids are described in Supplementary Figure S2 .
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