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Digestion of the PCR we did yesterday
Yesterday we amplified flhD, flhC, flhDC with its promoter and RBS+
Before the digestion, we have to determine the DNA concentration of the templates.
Measurement of DNA concentration
We used the biophotometer. We put 5 µL of template DNA in 95 µL of water. The Blank was made with 5 µL of EB Buffer (the one that was used for the elution of the DNA) in 95 µL of water.
| Template
| Concentration
(µg/µL)
|
PCR 130
E0240 RBS +
| 0.44
|
PCR 131
flhD RBS -
| 0.06
|
PCR 132
flhC RBS -
| 0.12
|
PCR 133
flhDC with promoter
| 0.08
|
MP 142
pSB3K3
| 0.04
|
MP 122
pSB1A2
| 0.2
|
Digestion
Protocol
| Digestion name
| Template DNA
| Enzymes
| Volume of DNA
|
| D 140
| PCR 130 - E0240 RBS +
| PstI
| 5 µL
|
| D 141
| PCR 131 - flhD rbs -
| EcoRI-SpeI
| 5 µL
|
| D 142
| PCR 132 - flhC rbs -
| EcoRI-SpeI
| 5 µL
|
| D 143
| PCR 133 - flhDC + prom
| EcoRI-SpeI
| 5 µL
|
| D 144
| MP 142 - pSB3K3
| EcoRI-SpeI
| 5 µL
|
| D 145
| MP122 - pSB1A2
| EcoRI-SpeI
| 5 µL
|
- X µL of Template DNA
- Buffer (n°2) 10X : 3µL
- BSA 100X : 0.3µL
- Pure water qsp 30 µL
- 1 µL of each enzyme
- Incubate during about 3h at 37°C, then 20 minutes at 65°C (to inactivate the enzymes).
- Exclusively for D 144 : 2 µL of Antarctic Phosphatase was added, 30 min at 37°C then 5 min at 65°C.
Results of the digestion
 Result of the digestion to build the measurement plasmid Electrophoresis settings
- Gel : 1 % agar
- 4µL template DNA for D137
- All the digestion product for D138 because we have to separate two products, the backbone measures 2056 bp and the insert we want to extract is 899 bp long.
- 10µL QuickLoad DNA ladder 1 kb
| Name
| Band
| Expected size
| Measured size
|
| D 137
| 2
| 2727 bp
| 3000 bp
|
| D 138
| 3&4
| 899 bp
| ~1000 bp
|
Gel extraction and DNA purification
To extract the biobrick E0240 from the gel, we used the standard protocol number 8.
To purify the DNA we used the standard protocol number 10
The ligation will be done tomorrow.
- Protocol (see # 3) Experiments done by QIAcube
| Name
| Ligation
| Biobricks
| Description
|
| MP144.1
| L128.1
| pFlgA-RFP
D132 (FI) - D136 (FV)
|  
|
| MP144.2
| L128.2
|
| MP144.3
| L128.3
|
| MP144.4
| L128.4
|
| MP145.1
| L129.1
| pFlgB-RFP
D133 (FI) - D136 (FV)
|
|
| MP145.2
| L129.2
|
| MP145.6
| L129.6
|
| MP145.7
| L129.7
|
| MP146.1
| L130.1
| pFlhB-RFP
D134 (FI) - D136 (FV)
|
|
| MP146.2
| L130.2
|
| MP146.7
| L130.7
|
| MP146.8
| L130.8
|
Glycerol Stocks
| Strain
| Ligation
| Biobricks
| Description
|
| S143.1
| L128.1
| pFlgA-RFP
D132 (FI) - D136 (FV)
|
|
| S143.2
| L128.2
|
| S143.3
| L128.3
|
| S143.4
| L128.4
|
| S144.1
| L129.1
| pFlgB-RFP
D133 (FI) - D136 (FV)
|
|
| S144.2
| L129.2
|
| S144.6
| L129.6
|
| S144.7
| L129.7
|
| S145.1
| L130.1
| pFlhB-RFP
D134 (FI) - D136 (FV)
|
|
| S145.2
| L130.2
|
| S145.7
| L130.7
|
| S145.8
| L130.8
|
Results of the transformations we did yesterday
Your results please Cyprien!!!???
Concentration of the MiniPreps
| Miniprep
| Concentration (µg/mL)
| Ratio 260/280
|
| MP144.1
| 127
| 1.67
|
| MP144.2
| 166
| 1.66
|
| MP144.3
| 163
| 1.57
|
| MP144.4
| 154
| 1.63
|
| MP145.1
| 136
| 1.67
|
| MP145.2
| 151
| 1.63
|
| MP145.6
| 163
| 1.67
|
| MP145.7
| 188
| 1.57
|
| MP146.1
| 298
| 1.70
|
| MP146.2
| 142
| 1.64
|
| MP146.7
| 170
| 1.52
|
| MP146.8
| 143
| 1.55
|
New PCR screening with the right primers
Transformants with pFlgA, pFlgB and pFlhB cloned into J61002 are analysed by PCR but this time with the right primers: VF2 (O18) and VR (O19).
- template: bacteria from glycerol stock
- positive control: J61002 (with ptet-mRFP)
- negative control: no template
Electrophoresis
- 1% agarose gel
- 10 µL of each sample loaded
| well n°
| 1
| 2
| 3
| 4
| 5
| 6
| 7
| 8
| 9
| 10
| 11
| 12
| 13
| 14
| 15
| 16
|
| sample
| 1 kb DNA ladder
| positive control
| negative control
| L128 (pFlgA)
| L129 (pFlgB)
| L130 (pFlhB)
| 100 bp DNA ladder
|
| clone n°
|
| 1
| 2
| 3
| 4
| 1
| 2
| 6
| 7
| 1
| 2
| 7
| 8
|
|
| red fluorescence
|
| yes
| yes
| no
| no
| yes
| yes
| no
| no
| yes
| no
| no
| no
|
|
| expected size
|
| 1161 bp
| 0 bp
| 1339 bp
| 1339 bp
| 1338 bp
|
|
| measured size
|
| 1,2 kb
| 0 kb
| 1,2 kb
| 1,2 kb
| 1,3 kb
| 1,3 kb
| 1,2 kb
| 0 kb
| 1,4 kb
| 1,4 kb
| 1,2 kb
| 1,2 kb
| 1,2 kb
| 1,2 kb
|
|
|
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