For libraries 1 and 2, we digested the Lenti-gRNA-Puro plasmid with Esp3I (NEB). For library 2 in vivo experiments, we digested the Lenti-p3-eGFP plasmid. After digestion, the plasmid was dephosphorylated by rSAP (NEB), followed by gel extraction. The oligo-pool amplicons were assembled into the linearized plasmid using NEBuilder HiFi DNA Assembly Master Mix (NEB). The product was precipitated by adding one volume of Isopropanol (99%) and 0.02 volumes of 5M NaCl solution. The mix was vortexed for 10 sec and incubated at room temperature for 15 min, followed by 15 min centrifugation (17,000 × g). The supernatant was discarded and replaced by four volumes of ice-cold ethanol (80%). Ethanol was removed immediately, the wash was repeated once, and the pellet was air-dried. Next, the pellet was dissolved in H2O. In 15 (library 1) or 2 (library 2) transformation replicates, 100 ng of plasmid library were transformed per 25 µl of Endura Competent Cells (Lucigen) using a Gene Pulser II device (Bio-Rad). Transformed cells were recovered in S.O.C. media for 1 h at 30 °C. Transformed cells were then spread on LB agar plates containing 100 µg/mL ampicillin. After incubation at 30 °C for 16 h, the colonies were scraped, and plasmids were purified using a Plasmid Maxi Kit (Qiagen).
Gene pulser 2 device
Manufactured by Bio-Rad
The Gene Pulser II device is a laboratory instrument used for electroporation, a technique that introduces exogenous molecules, such as DNA, into cells by creating transient pores in the cell membrane using electrical pulses. The device allows users to precisely control the electrical parameters, including voltage, capacitance, and pulse duration, to optimize the electroporation conditions for various cell types and applications.
Automatically generated - may contain errors
2 protocols using gene pulser 2 device
1
Lenti-Plasmid Library Construction
2
Lenti-Plasmid Library Construction
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For libraries 1 and 2, we digested the Lenti-gRNA-Puro plasmid with Esp3I (NEB). For library 2 in vivo experiments, we digested the Lenti-p3-eGFP plasmid. After digestion, the plasmid was dephosphorylated by rSAP (NEB), followed by gel extraction. The oligo-pool amplicons were assembled into the linearized plasmid using NEBuilder HiFi DNA Assembly Master Mix (NEB). The product was precipitated by adding one volume of Isopropanol (99%) and 0.02 volumes of 5M NaCl solution. The mix was vortexed for 10 sec and incubated at room temperature for 15 min, followed by 15 min centrifugation (17,000 × g). The supernatant was discarded and replaced by four volumes of ice-cold ethanol (80%). Ethanol was removed immediately, the wash was repeated once, and the pellet was air-dried. Next, the pellet was dissolved in H2O. In 15 (library 1) or 2 (library 2) transformation replicates, 100 ng of plasmid library were transformed per 25 µl of Endura Competent Cells (Lucigen) using a Gene Pulser II device (Bio-Rad). Transformed cells were recovered in S.O.C. media for 1 h at 30 °C. Transformed cells were then spread on LB agar plates containing 100 µg/mL ampicillin. After incubation at 30 °C for 16 h, the colonies were scraped, and plasmids were purified using a Plasmid Maxi Kit (Qiagen).
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