Enriched cosmids identified by NGS were retrieved by transformation of cosmid extracts in E. coli. Cosmids were then identified through random screening and Sanger sequencing. Following isolation, individual cosmids were transfected into L. infantum for testing their resistance against TBF. Genes of interest were amplified from L. infantum genomic DNA and cloned into the Leishmania expression vector pSP72αHYGROα. Each plasmid insert was sequenced to confirm the absence of mutation. A total of 20 μg of plasmid or cosmid DNA were transfected into Leishmania promastigotes by electroporation using a BioRad GenePulse XCell Electroporation System with one pulse at 450 V and capacitance at 500 μF. Transfected parasites were selected and maintained with hygromycin (600 μg/ml).
Genepulse xcell electroporation system
Manufactured by Bio-Rad
The GenePulse XCell Electroporation System is a laboratory instrument designed for the delivery of nucleic acids, such as DNA or RNA, into cells through the process of electroporation. It provides a controlled electrical pulse to temporarily open pores in the cell membrane, allowing the passage of genetic material into the cells.
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Lab products found in correlation
3 protocols using genepulse xcell electroporation system
1
Cosmid-based Leishmania Drug Resistance
2
CRISPR-Cas9 Gene Editing in PAH-Mutant COS-7 Cells
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Electroporation was performed according to the instructions provided with the Gene Pulse Xcell electroporation system (Bio-Rad). Prior to electroporation, PAH_c.1222C>T COS-7 cells were washed with PBS and resuspended in Gene Pulser electroporation buffer (Bio-Rad) at 2.5 × 106 cells/mL. Ten micrograms of pRSI9-1222sgRNA plasmid, 10 μg FokI-dCas9-zsGreen plasmid and 0.1 nM to 1 nM ssODN were added to the 0.4 cm cuvette with 400 μL cells and mixed gently. The optimized electroporation setting for PAH_c.1222C>T COS-7 cells was previously determined to be the following: pulse type (square wave), V (200 V), PL (15 msec) and cuvette (0.4 cm). After electroporation, 400 μL of the electroporated cells were transferred into 6-well plates. These electroporated cells were cultured in complete growth medium with or without 15 μM RS-1 (Sigma-Aldrich) at 37 °C for 96 hours.
3
Establishing Genotype 2a HCV Replicons
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Genotype 2a (JFH-1) full genomic and subgenomic replicons were provided courtesy of Professor Takaji Wakita (Tokyo, Japan). The constructs were electroporated into naive cells using the BioRad Gene PulseXcell electroporation system and then selected using G418 supplemented media for approximately three weeks. The G418-resistant colonies were then pooled and cultured on 10cm dishes. HCV levels were determined using RNA and protein isolation from cell lysates.
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