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Team:Paris Saclay/Notebook/July/1
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==='''A - Aerobic/Anaerobic regulation system'''=== | ==='''A - Aerobic/Anaerobic regulation system'''=== | ||
| - | ===='''Objective : obtaining | + | ===='''Objective : obtaining BBa_K1155000'''==== |
| - | ===='''1 - Digestion of | + | ===='''1 - Digestion of pSB1C3 plasmid and PCR products : Pndh* by EcoRI/ PstI'''==== |
Zhou | Zhou | ||
| + | |||
| + | We did a PCR amplification. The products were good. After we made digestion by EcoRI/PstI. | ||
Used quantities : | Used quantities : | ||
| - | * | + | * pSB1C3 : |
** Plasmid : 4µL | ** Plasmid : 4µL | ||
** EcoRI FD : 0.5µL | ** EcoRI FD : 0.5µL | ||
| Line 21: | Line 23: | ||
** H2O : 2.2µL | ** H2O : 2.2µL | ||
| - | * | + | * Pndh* : |
| - | ** | + | ** Pndh* : 20µL |
** EcoRI FD : 0.75µL | ** EcoRI FD : 0.75µL | ||
** PstI FD : 0.75µL | ** PstI FD : 0.75µL | ||
| Line 28: | Line 30: | ||
** H2O : 5.5µL | ** H2O : 5.5µL | ||
| - | We | + | We incubate the digestion at 37°C during 3 hours. |
| - | ===='''2 - Ligation of | + | ===='''2 - Ligation of pSB1C3 and Pndh*'''==== |
Sheng, Zhou | Sheng, Zhou | ||
| Line 36: | Line 38: | ||
Used quantities : | Used quantities : | ||
* Mix A : we mix our digestion mixes : | * Mix A : we mix our digestion mixes : | ||
| - | ** Digestion mix of | + | ** Digestion mix of pSB1C3 : 4µL |
| - | ** Digestion mix of | + | ** Digestion mix of Pndh* : 30µL |
** Buffer ligation : 2µL | ** Buffer ligation : 2µL | ||
** H2O : 14µL | ** H2O : 14µL | ||
| + | Incubate the ligation at 37oC for 1 hour. | ||
| - | * Then, we | + | * Then, we inactivate EcoRI/SpeI activity by ethanol precipitation. |
Protocol : [[Team:Paris_Saclay/ethanol|Ethanol precipitation]] | Protocol : [[Team:Paris_Saclay/ethanol|Ethanol precipitation]] | ||
| Line 54: | Line 57: | ||
** H2O : 15µL | ** H2O : 15µL | ||
| - | We | + | We incubate the ligation at 37oC for 1h30. |
==='''B - PCB sensor system'''=== | ==='''B - PCB sensor system'''=== | ||
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Latest revision as of 00:48, 5 October 2013
Contents |
Notebook : July 1
Lab work
A - Aerobic/Anaerobic regulation system
Objective : obtaining BBa_K1155000
1 - Digestion of pSB1C3 plasmid and PCR products : Pndh* by EcoRI/ PstI
Zhou
We did a PCR amplification. The products were good. After we made digestion by EcoRI/PstI.
Used quantities :
- pSB1C3 :
- Plasmid : 4µL
- EcoRI FD : 0.5µL
- PstI FD : 0.5µL
- Buffer FD : 0.8µL
- H2O : 2.2µL
- Pndh* :
- Pndh* : 20µL
- EcoRI FD : 0.75µL
- PstI FD : 0.75µL
- Buffer FD : 3µL
- H2O : 5.5µL
We incubate the digestion at 37°C during 3 hours.
2 - Ligation of pSB1C3 and Pndh*
Sheng, Zhou
Used quantities :
- Mix A : we mix our digestion mixes :
- Digestion mix of pSB1C3 : 4µL
- Digestion mix of Pndh* : 30µL
- Buffer ligation : 2µL
- H2O : 14µL
Incubate the ligation at 37oC for 1 hour.
- Then, we inactivate EcoRI/SpeI activity by ethanol precipitation.
Protocol : Ethanol precipitation
We used 50µL of DNA. At the end, we dilute our DNA in 20µL of H2O.
- Finally we did the ligation mix :
- Buffer ligation : 2µL
- Ligase : 1µL
- DNA : 2µL
- H2O : 15µL
We incubate the ligation at 37oC for 1h30.
B - PCB sensor system
Objective : obtaining BphR2 protein
1 - Design of oligos for amplification of BphR2 gene of Pseudomonas pseudoalcaligenes, Pseudomonas oleovorans
Abdou, Sheng, Zhou
We used software gene manager to find the correct oligopeptides for amplification of BphR2 genes in Pseudomonas pseudoalcaligenes, Pseudomonas oleovorans.
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