IGEM:University of East Anglia (UEA), Norwich, UK/2009/Notebook/NRP-UEA-Norwich iGEM/2014/07/10

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Week 4 Day 4


- Agrobacterium-mediated transient expression in leaves of N. benthamiana PROTOCOL:

1. Single colony of each constructs 2,3,4,10,11 grown overnight at 28 °C in 5 ml media containing chloramphenicol.

2. Pellet by centrifugation at 4000 rpm for 15 min.

3. Resuspend in 10 mM MES, 10 mM MgCl2 and 150 μM Acetosyringone.

4. 1:100 dilution to give a final OD of 0.1-0.5

5. Incubate at RT for 4 hours.

6. Create a small puncture using a needle on the underside of N. benthamiana leaves of plants 6-8 weeks old. Using a blunt syringe infiltrate the agro constructs into the leaf puncture. Each construct is infiltrated into 4 sections on one leaf. 2 constructs per plant.

7. Store plants in controlled environment at 19-23 °C for 3 days.

-Ligation reaction to join PSB1C3 and GG compatible ends •PCR program? •Transfored into E.coli •Spread plated onto CAM plates