Aleksandra
Glycerols of the yesterday's overnight cultures
15%glycerol (375µl 60%glycerol + 1125µl cultute), 2 for each (different colonies):
- attC/term/attC/mRFP
- weak RBS/TetA(C)/LuxI/term
- medium RBS/TetA(C)/LuxI/term
- strong RBS/TetA(C)/LuxI/term
- weak RBS/LacZ alpha/LuxI/term
- strong RBS/LacZ alpha/LuxI/term
Minipreps of the yesterday's overnight cultures, 2 for each part(cultures starting from colonies #1 and #2 on each plate)
Elution in 35µl EB buffer
- attC/term/attC/mRFP
- weak RBS/TetA(C)/LuxI/term
- medium RBS/TetA(C)/LuxI/term
- strong RBS/TetA(C)/LuxI/term
- weak RBS/LacZ alpha/LuxI/term
- strong RBS/LacZ alpha/LuxI/term
Quantification of the DNA in the minipreps by Nanodrop
Sample name |
260/280 |
260/230 |
ng/µl
|
|
|
|
|
|
|
|
|
attC/term/attC/mRFP 1 |
1.97 |
2.11 |
159.6
|
attC/term/attC/mRFP 2 |
1.96 |
1.87 |
195.3
|
weakRBS/TetA(C)/LuxI/term 1 |
1.96 |
2.28 |
381.3
|
weakRBS/TetA(C)/LuxI/term 2 |
1.96 |
2.30 |
291.9
|
mediumRBS/TetA(C)/LuxI/term 1 |
1.96 |
2.26 |
353.2
|
mediumRBS/TetA(C)/LuxI/term 2 |
1.95 |
2.28 |
490.1
|
standardRBS/TetA(C)/LuxI/term 1 |
1.96 |
2.27 |
425.2
|
standardRBS/TetA(C)/LuxI/term 2 |
1.96 |
2.31 |
362.6
|
weakRBS/LacZ alpha/LuxI/term 1 |
1.96 |
2.20 |
260.2
|
weakRBS/LacZ alpha/LuxI/term 2 |
1.97 |
2.28 |
273.3
|
standardRBS/LacZ alpha/LuxI/term 1 |
1.98 |
2.24 |
248.6
|
standardRBS/LacZ alpha/LuxI/term 2 |
1.97 |
2.23 |
265.0
|
Restriction digests
Preparative digests (20µl miniprep + 2x1µl enzymes + 2.5µl buffer4 + 0.25µl BSA)
Minipreps from today (only with minipreps from colony #1):
- attC/term/attC/mRFP with EcoRI-HF+XbaI
- weak RBS/TetA(C)/LuxI/term with EcoRI-HF+SpeI
- medium RBS/TetA(C)/LuxI/term with EcoRI-HF+SpeI
- strong RBS/TetA(C)/LuxI/term with EcoRI-HF+SpeI
- weak RBS/LacZ alpha/LuxI/term with EcoRI-HF+SpeI
- strong RBS/LacZ alpha/LuxI/term with EcoRI-HF+SpeI
Minipreps from 29/07:
- pLux/GFP with EcoRI-HF+XbaI
- Ps/weakRBS/LuxR with EcoRI-HF+SpeI
- Pm/weakRBS/LuxR with EcoRI-HF+SpeI
- Ps/mediumRBS/LuxR with EcoRI-HF+SpeI
- Pm/mediumRBS/LuxR with EcoRI-HF+SpeI
- Ps/stongRBS/LuxR with EcoRI-HF+SpeI
- Pm/strongRBS/LuxR with EcoRI-HF+SpeI
Verification digests (5µl miniprep + 12µl water + 2x1µl enzymes + 2µl buffer4 + 0.2µl BSA)
- attC/term/attC/mRFP (miniprep from colony #1) with EcoRI-HF+PstI
- attC/term/attC/mRFP (miniprep from colony #2) with EcoRI-HF+PstI
Gel electrophoresis (1.5%agarose w/v, 50V)
20µl restriction digest+5µl 6x loading buffer
weak RBS/TetA(C)/LuxI/term with EcoRI-HF+SpeI
- weak RBS/TetA(C)/LuxI/term with EcoRI-HF+SpeI
- ladder
- medium RBS/TetA(C)/LuxI/term with EcoRI-HF+SpeI
- -
- strong RBS/TetA(C)/LuxI/term with EcoRI-HF+SpeI
- -
- weak RBS/LacZ alpha/LuxI/term with EcoRI-HF+SpeI
- -
- strong RBS/LacZ alpha/LuxI/term with EcoRI-HF+SpeI
- -
- attC/term/attC/mRFP #1 (digested with EcoRI-HF+PstI for verification)
- attC/term/attC/mRFP #2 (digested with EcoRI-HF+PstI for verification)
- Ps/weakRBS/LuxR with EcoRI-HF+SpeI
- -
- Pm/weakRBS/LuxR with EcoRI-HF+SpeI
- -
- Ps/mediumRBS/LuxR with EcoRI-HF+SpeI
- -
- Pm/mediumRBS/LuxR with EcoRI-HF+SpeI
- -
- Ps/stongRBS/LuxR with EcoRI-HF+SpeI
- -
- Pm/strongRBS/LuxR with EcoRI-HF+SpeI
- ladder
Gel extraction
Elution in 35µl warm water
- weak RBS/TetA(C)/LuxI/term
- medium RBS/TetA(C)/LuxI/term
- strong RBS/TetA(C)/LuxI/term
- weak RBS/LacZ alpha/LuxI/term
- strong RBS/LacZ alpha/LuxI/term
- Ps/weakRBS/LuxR
- Pm/weakRBS/LuxR
- Ps/mediumRBS/LuxR
- Pm/mediumRBS/LuxR
- Pm/strongRBS/LuxR
DNA quantification after gel extraction. Measured with the Nanodrop
Sample name |
260/280 |
260/230 |
ng/µl
|
|
|
|
|
|
|
|
|
weakRBS/TetA(C)/LuxI/term 1 |
1.77 |
0.13 |
27.1
|
mediumRBS/TetA(C)/LuxI/term 1 |
1.68 |
0.10 |
23.2
|
standardRBS/TetA(C)/LuxI/term 1 |
1.77 |
0.16 |
27.3
|
weakRBS/LacZ alpha/LuxI/term 1 |
1.77 |
0.02 |
8.1
|
standardRBS/LacZ alpha/LuxI/term 1 |
1.59 |
0.03 |
9.3
|
Pm/standRBS/LuxR |
1.90 |
0.01 |
3.5*
|
Pm/medRBS/LuxR |
1.22 |
0.02 |
1.5*
|
Ps/medRBS/LuxR |
2.20 |
0.03 |
2.5
|
Pm/weakRBS/LuxR |
1.06 |
0.01 |
2.3*
|
Ps/weakRBS/LuxR |
1.55 |
0.06 |
7.6
|
*Apparently no DNA in the sample : no peak on the Nanodrop.
PCR purification kit
- pLux/GFP
- attC/term/attC/mRFP (digested with EcoRI-HF and Xba-I for future ligation)
DNA quantification after PCR purification kit. Measured with the Nanodrop
Sample name |
260/280 |
260/230 |
ng/µl
|
|
|
|
|
|
|
|
|
attC/term/attC/mRFP (from colony#1) |
1.52 |
0.59 |
8.8
|
attC/term/attC/mRFP (from colony#2) |
1.69 |
0.08 |
9.8
|
pLux/GFP (A from 29/07) |
1.69 |
1.36 |
77.3
|
Ligation at 16°C overnight
- 1µl pLux/GFP + 6µl stand.RBS/TetA(C)/LuxI/term + 1µl buffer + 1.5µl water + 0.5µl ligase
- 1µl pLux/GFP + 6µl med.RBS/TetA(C)/LuxI/term + 1µl buffer + 1.5µl water + 0.5µl ligase
- 1µl pLux/GFP + 6µl weak.RBS/TetA(C)/LuxI/term + 1µl buffer + 1.5µl water + 0.5µl ligase
- 1µl pLux/GFP + 16.5µl stand.RBS/LacZα/LuxI/term + 2µl buffer + 0.5µl ligase
- 1µl pLux/GFP + 16.5µl weakRBS/LacZα/LuxI/term + 2µl buffer + 0.5µl ligase
- control 1 : 1µl pLux/GFP + 1µl buffer + 7.5µl water + 0.5µl ligase
- control 2 : 6µl stand.RBS/TetA(C)/LuxI/term + 1µl buffer + 2.5µl water + 0.5µl ligase
Overnight cultures
- 12ml cultures LB+Kan started from 16 different colonies on the attC/term/attC/mRFP transformation plates from yesterday
- 10ml culture LB+Kan of attC (from glycerol)
- 10ml culture LB+Kan of term/attC/mRFP (from glycerol)
|