IGEM:University of East Anglia (UEA), Norwich, UK/2009/Notebook/NRP-UEA-Norwich iGEM/2014/07/10: Difference between revisions
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Jessica Gray (talk | contribs) (Autocreate 2014/07/10 Entry for IGEM:University_of_East_Anglia_(UEA),_Norwich,_UK/2009/Notebook/NRP-UEA-Norwich_iGEM) |
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== | ==Week 4 Day 4== | ||
TSL | |||
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- 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. | |||
Revision as of 03:33, 14 July 2014
 iGEM Project name 1
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Week 4 Day 4TSL - 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.
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