Electronic Journal of Biotechnology ISSN: 0717-3458
  © 2007 by Universidad Católica de Valparaíso -- Chile
Vol. 10 No. 2, Issue of April 15, 2007
 

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 28C 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 28C with shake

4

 Centrifuge the bacteria (5000 rpm for 10 min)
 Resusped in groinfiltration solution
 10 mM MgC12; 10 mM MES and 100 M 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 28C, 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

1a. Gene silencing of transiently expressed sequences.
1b. Triggering gene silencing of established transgene by transient expression.
1c. Virus-induced gene silencing (VIGS).
2. Systemic gene silencing.
3a. Suppressing gene silencing of transiently expressed sequences.
3b. Silencing of transgenes or endogenous genes cannot be established in the presence of silencing suppressor.


Supported by UNESCO / MIRCEN network