pLEXSY-hyg-2-hAm

Protocol tips

Upstream tips	Protocol tips	Downstream tips
	108 cells of I.L.L. (22) were washed by electroporation buffer (eppendorf, Germany) and suspended in 1 ml electroporation buffer. The recombinant pLEXSY-hyg2-hAm linearized by SwaI (Ferments, Lithuania). Then 50μl of linearized DNA containing 5μgr DNA was added to 450 μl of cell suspension containing 108 cell/min a 4 mm cuvette. The I.L.L. cells were transfected by using two pulse 2000V (interval: 10S) in a Multiporator (eppendorf, Germany) (24). Stable transfectants were selected on LB broth media containing 100 μg/ml hygromycin (sigma).

Protocol tips

108 cells of I.L.L. (22) were washed by electroporation buffer (eppendorf, Germany) and suspended in 1 ml electroporation buffer. The recombinant pLEXSY-hyg2-hAm linearized by SwaI (Ferments, Lithuania). Then 50μl of linearized DNA containing 5μgr DNA was added to 450 μl of cell suspension containing 108 cell/min a 4 mm cuvette. The I.L.L. cells were transfected by using two pulse 2000V (interval: 10S) in a Multiporator (eppendorf, Germany) (24). Stable transfectants were selected on LB broth media containing 100 μg/ml hygromycin (sigma).

Publication protocol

108 cells of I.L.L. (22) were washed by electroporation buffer (eppendorf, Germany) and suspended in 1 ml electroporation buffer. The recombinant pLEXSY-hyg2-hAm linearized by SwaI (Ferments, Lithuania). Then 50μl of linearized DNA containing 5μgr DNA was added to 450 μl of cell suspension containing 108 cell/min a 4 mm cuvette. The I.L.L. cells were transfected by using two pulse 2000V (interval: 10S) in a Multiporator (eppendorf, Germany) (24). Stable transfectants were selected on LB broth media containing 100 μg/ml hygromycin (sigma). Integration of the expression cassette into the small subunit rRNA (ssu) locus of I.L.L. was confirmed by diagnostic PCR reaction by using F3001 forward primer (located on ssu locus of I.L.L. genome) and hAm reverse primer (Table 1).

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Manufacturer protocol

Download the product protocol from Mojgan BANDEHPOUR, Dept. of Biotechnology, School of Medicine, for pLEXSY-hyg-2-hAm below.

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