pJL TRBO-G

Protocol tips

Upstream tips	Protocol tips	Downstream tips
	A. tumefaciens GV3101 carrying either pJL TRBO-G or pCBNoX P19, were grown in 4 ml LB medium for 24 h at 28 °C and shaking at 250 rpm. The cultures were then transferred into 100 ml LB medium having 200 μM of acetosyringone (Sigma-Aldrich) grown overnight at 28 °C and shaking at 250 rpm.	Cells were harvested by centrifugation at 3000 g for 10 min and re-suspended in infiltration buffer (pH 5.6, 10 mM MES, 10 mM MgCl2 and 200 μM acetosyringone) to achieve an OD600 of 0.4. The pJL TRBO-G expression vector was mixed in a 1:1 volume ratio with the gene-silencing suppressor (pCBNoX P19). The mixed Agrobacterium suspensions were incubated in the dark at room temperature for 2–3 h before infiltration. The incubated Agrobacterium suspensions were infiltrated into the abaxial surface of leaves using a 1-ml syringe without needle. The agroinfiltrated plants were incubated in the growth chamber for 4–8 days after which the leaves were harvested.

Protocol tips
A. tumefaciens GV3101 carrying either pJL TRBO-G or pCBNoX P19, were grown in 4 ml LB medium for 24 h at 28 °C and shaking at 250 rpm. The cultures were then transferred into 100 ml LB medium having 200 μM of acetosyringone (Sigma-Aldrich) grown overnight at 28 °C and shaking at 250 rpm.
Downstream tips
Cells were harvested by centrifugation at 3000 g for 10 min and re-suspended in infiltration buffer (pH 5.6, 10 mM MES, 10 mM MgCl2 and 200 μM acetosyringone) to achieve an OD600 of 0.4. The pJL TRBO-G expression vector was mixed in a 1:1 volume ratio with the gene-silencing suppressor (pCBNoX P19). The mixed Agrobacterium suspensions were incubated in the dark at room temperature for 2–3 h before infiltration. The incubated Agrobacterium suspensions were infiltrated into the abaxial surface of leaves using a 1-ml syringe without needle. The agroinfiltrated plants were incubated in the growth chamber for 4–8 days after which the leaves were harvested.

Publication protocol

A. tumefaciens GV3101 carrying either pJL TRBO-G or pCBNoX P19, were grown in 4 ml LB medium for 24 h at 28 °C and shaking at 250 rpm. The cultures were then transferred into 100 ml LB medium having 200 μM of acetosyringone (Sigma-Aldrich) grown overnight at 28 °C and shaking at 250 rpm. Cells were harvested by centrifugation at 3000 g for 10 min and re-suspended in infiltration buffer (pH 5.6, 10 mM MES, 10 mM MgCl2 and 200 μM acetosyringone) to achieve an OD600 of 0.4. The pJL TRBO-G expression vector was mixed in a 1:1 volume ratio with the gene-silencing suppressor (pCBNoX P19). The mixed Agrobacterium suspensions were incubated in the dark at room temperature for 2–3 h before infiltration. The incubated Agrobacterium suspensions were infiltrated into the abaxial surface of leaves using a 1-ml syringe without needle. The agroinfiltrated plants were incubated in the growth chamber for 4–8 days after which the leaves were harvested.

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