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Team:Evry/Notebook/w12
From 2013.igem.org
Week 12: 2nd September - 8th September
Creation of a E. Coli ΔFur
02/09/13 04/09/13 We picked colonies from our four plates, that we grew overnight at 42°C, to plate each of them on one plate with kanamycin
TECAN
02/09/13 To further improve the interpretability of the plate reading, we need to transfer our construction from a BL21 to a TOP10 strain. As a start, we transformed the following constructions:
- AceB with LacI
- FecA with GFP
- FepA with GFP
- FepA with LacI
- Fes with GFP
- Fes with LacI
- YbiL with GFP
- YbiL with LacI
- YncE with GFP
- YncE with LacI
- control positive with pSB1A3
- control negative
We did not have anymore AceB with GFP plasmid available to realize the transformation. We pre-cultured a remaining glycerol stock at 37°C overnight:
10 mL LB
10 µL Carbenicillin 1000X
50 µL cells from glycerol stock
03/09/13
Plasmid 3
We launch another PC to get Enterobactins A, B, C, D, E and F genes. Add the recquired primers in each tube:- EntA: primers 065 and 066
- EntB: primers 067 and 068
- EntC: primers 069 and 070
- EntD: primers 071 and 072
- EntE: primers 073 and 074
- EntF: primers 075 and 076
- Positive controle: primers 009 and 010
- Negative controle: primers 009 and 010
For more details about our primers, see the corresponding page.
We then prepared a master mix 1 with:
- 9 x 27,5 = 247,5 µL of distilled water
- 9 x 1 = 9 µL of dNTPs
- 9 x 10 = 90 µL of Q
5 Buffer - 9 x 0,5 = 4,5 µL of Q
5 For the negative controle, we added 1 µL of distilled water in tube 8 and 39 µL of master mix 1.
For other tubes (6 genes and positive controle), we added 8 µL of genomic DNA in the master mix 1. Then add 40 µL of master mix 1' in each tube.
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