Table 1 - A practical approach to the understanding and teaching of RNA silencing in plants

Table 1. Schedule for all the silencing experiments.

Days
1	Plate the agrobacterium in LB-Agar plate with appropriate antibiotic
	1a, 1b, 2, 3a, 3b			35-ds GFP		Kan50mg/ml; Genta 40mg/ml and Rif 100mg/ml
	1a, 3a			35-GFP		Kan50mg/ml; Genta 40mg/ml and Rif 100mg/ml
	3a, 3b			35-Hc-PRO		Kan50mg/ml; Genta 40mg/ml and Rif 100mg/ml
	1c			TRV1		Kan50mg/ml; Genta 40mg/ml and Rif 100mg/ml
	1c			TRV2		Kan50mg/ml; Genta 40mg/ml and Rif 100mg/ml
	At this time the plants should have the proper size to be agroinfiltrated (Figure 4)
2	Pick a colony up and inoculate 5 ml with the appropriate antibiotic Incubate at 28ºC with shaker
3	Used 0.5 ml of the overnight culture to inoculate 20 ml of LB media Correct antibiotic and Acetosyringone (20 mM final) Incubate at 28ºC with shake
4	Centrifuge the bacteria (5000 rpm for 10 min) Resusped in groinfiltration solution 10 mM MgC12; 10 mM MES and 100 µM Acetosyringone Quantify the OD (600) of the bacterias and make the appropriate dilution
	1a, 1b, 2 1a 1a 1c 1c		35-ds GFP 35-GFP 35-Hc-PRO TRV1 TRV2		1 OD final 0.5 OD final 0.5 OD final 1 OD final 1 OD final
	Leave the bacteria solution at room temperature for 2 to 4 hrs Infiltrate the underside leaf with a 2 or 10 ml syringe. See Movie 1 Maintain the plants in growth chambers at NO more than 28ºC, 14 hrs light cycle
6 to 10	Silencing efects should be seen 2 to 10 days post the infiltrations days.
	1a, 3a 1b, 3b 1c, 2 3c	2 days should be enough but 4 or 5 days in better 5 days should be enough 1 week should be enough see light effects and 2 to 4 weeks to see strong ones 2 weeks should be enough