pIRES2-EGFP-PBD-1

Protocol tips

Upstream tips	Protocol tips	Downstream tips
	Lipofectamine 2000 reagent (Invitrogen) was adopted for gene transfection using the provided protocol. The Marc 145 cells were seeded in 96-well plates at the density of 1 × 104 cells/100 µL for each well. Twenty-four h later (at a confluency of about 80–85 %), the cells were transfected by Lipofectamine 2000 with the pIRES2-EGFP-PBD-1 plasmid, and the ratio of plasmid mass to lipofectamine 2000 volume was 1:3. The transiently transfected cells were grown first in growth medium supplemented with 800 µg/mL G418 for selecting stably transfected clones.

Protocol tips

Lipofectamine 2000 reagent (Invitrogen) was adopted for gene transfection using the provided protocol. The Marc 145 cells were seeded in 96-well plates at the density of 1 × 104 cells/100 µL for each well. Twenty-four h later (at a confluency of about 80–85 %), the cells were transfected by Lipofectamine 2000 with the pIRES2-EGFP-PBD-1 plasmid, and the ratio of plasmid mass to lipofectamine 2000 volume was 1:3. The transiently transfected cells were grown first in growth medium supplemented with 800 µg/mL G418 for selecting stably transfected clones.

Publication protocol

Lipofectamine 2000 reagent (Invitrogen) was adopted for gene transfection using the provided protocol. The Marc 145 cells were seeded in 96-well plates at the density of 1 × 104 cells/100 µL for each well. Twenty-four h later (at a confluency of about 80–85 %), the cells were transfected by Lipofectamine 2000 with the pIRES2-EGFP-PBD-1 plasmid, and the ratio of plasmid mass to lipofectamine 2000 volume was 1:3. The transiently transfected cells were grown first in growth medium supplemented with 800 µg/mL G418 for selecting stably transfected clones. Non-transfected cells lack of the resistance to this antibiotic. One week later, the selection pressure was increased to 1,000 µg/mL G418. The transfected Marc 145 cells were transferred to 96- well plates at an average density of less than one cell per well in 100 µL of selection medium for screening high-producing clones, (procedure of “limited dilution” (Puck 1958). The basis for this method is a dilution of the cell suspension to a point were statistically less than one cell per well (here 0.9) are plated. Wells were checked for cell number and those containing a number of cell (≠ 1) were excluded for the subsequent measurements. In our hands the method was faster and easier than isolation of the clones by using cloning cylinder (Freimark et al. 2010). Following dilution, the cells were further incubated for 2–3 weeks. A total of 20 plates were screened in this study.

Full paper   Login or join for free to view the full paper.

Reviews

Discussion

Papers

Check out relevant papers found by Labettor's AI that are relevant for performing Protein Expression Prokaryotic cells - E. coli PBD1-EGFP using pIRES2-EGFP-PBD-1 from Hai-Jun Huang, Department of Animal Biotechnology and Cell Engin.

Manufacturer protocol

Download the product protocol from Hai-Jun Huang, Department of Animal Biotechnology and Cell Engin for pIRES2-EGFP-PBD-1 below.

Videos

Check out videos that might be relevant for performing Protein Expression Prokaryotic cells - E. coli PBD1-EGFP using pIRES2-EGFP-PBD-1 from Hai-Jun Huang, Department of Animal Biotechnology and Cell Engin. Please note that these videos are representative and steps or experiment specific processes must be kept in mind to expect desired results.