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Antoine
Résults transformation biobrick R0040/C0040/R0011/C0012
Positif for all
Minipreps
pBM43
From 10 ml of overnight culture to 60μL Vf.
electrophoresis Minipreps
5μL of each in wells.
3μL of 1kb Ladder
picture of the gel
Overnight culture
(biobrick R0040/C0040/R0011/C0012 in LB + Amp)*2 (one for glycerol)
- P1003 LB+Amp+Kan
- YejF KO (from KEIO collection , KanR disrupt YejF gene) (LB+ Kan)*2 (one for glycerol)
Aleksandra, Stéphane, Raphaël
Minipreps : 60 minipreps in total, 4 minipreps of a 5ml culture per biobrick, eluted with 50μL buffer and collected into 1 tube for a total volume of around 120μL:
- I732006 (LacZ)
- B0031 (weak RBS)
- B0032 (medium RBS)
- B0034 (standard RBS)
- J31007 (TetA C)
- C0062 (LuxR w/o RBS)
- J37033 (RBS/LuxR) from the 2007 distribution plate
- 23119 (Ps)
- J23110 (Pm)
- attC
- term/attC/mRFP
- B0015 (term)
- E0240 (RBS/GFP/term)
- K081008 (LuxI)
- R0062 (pLux)
Nanodrop to determine the amount of DNA in the miniprep samples:
| Sample name |
260/280 |
260/230 |
ng/μL
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| term (B0015) |
1.82 |
2.34 |
119.9
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| attC |
1.72 |
2.21 |
26.8
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| RBS/LuxI (K081008) |
1.85 |
2.35 |
102.3
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| Pmed (J23110) |
1.87 |
2.34 |
194.2
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| Pstr (J23119) |
1.86 |
2.31 |
169.5
|
| LuxR (C0062) |
1.86 |
2.41 |
188.0
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| TetA(C) (J31007) |
1.86 |
2.35 |
149.1
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| RBS/LuxR (J37033) |
1.81 |
2.28 |
114.4
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| stand.RBS (B0034) |
1.86 |
2.30 |
145.2
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| med.RBS (B0032) |
1.90 |
2.34 |
120.1
|
| weakRBS (B0031) |
1.86 |
2.32 |
163.2
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| LacZalpha (I732006) |
1.86 |
2.42 |
221.3
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| pLux (R0062) |
1.93 |
2.27 |
66.6
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| t/attC/mRFP |
1.97 |
2.13 |
33.1
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| RBS/GFP/term (E0240) |
1.89 |
2.39 |
120.6
Restriction digest
This time we use the protocol that says we should use around 3μg of DNA in a minimum amount of water. The volume of the miniprep to use is calculated acording to the nanodrop results. Digestion at 37°C for 2 hours.
- I732006 (LacZ): 25μL miniprep + 3μL buffer 10x (B4) + 0.3μL BSA 100x + 1μL EcoRI-HF + 1μL XbaI
- B0031 (weak RBS), in a double quantity for 2 constructs: 67μL miniprep + 8μL buffer 10x (B2) + 0.8μL BSA 100x + 2μL SpeI + 2μL PstI
- B0032 (medium RBS), in double: 76μL miniprep + 9μL buffer 10x (B2) + 0.9μL BSA 100x + 2μL SpeI + 2μL PstI
- B0034 (standard RBS), in double: 67μL miniprep + 8μL buffer 10x (B2) + 0.8μL BSA 100x + 2μL SpeI + 2μL PstI
- J31007 (TetA C): 25μL miniprep + 3μL buffer 10x (B3) + 0.3μL BSA 100x + 1μL XbaI + 1μL PstI
- C0062 (LuxR w/o RBS), in triple: 58μL miniprep + 7μL buffer 10x (B3) + 0.7μL BSA 100x + 2μL XbaI + 2μL PstI
- J37033 (RBS/LuxR) from 2007, in double : 50μL miniprep + 6μL buffer 10x (B3) + 0.6μL BSA 100x + 2μL XbaI + 2μL PstI
- 23119 (Ps)
- J23110 (Pm)
- attC
- term/attC/mRFP
- B0015 (term)
- E0240 (RBS/GFP/term)
- K081008 (LuxI)
- R0062 (pLux)
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Population counter
