"
Team:Paris Saclay/Notebook/July/9
From 2013.igem.org
(Difference between revisions)
(→2 - Digestion PCR products : BphR2, BphR1, BphA1 and digestion of PSB1C3) |
(→2 - Digestion PCR products : BphR2, BphR1, BphA1 and digestion of pSB1C3) |
||
| (5 intermediate revisions not shown) | |||
| Line 22: | Line 22: | ||
Zhou | Zhou | ||
| - | BphR1, BphA1 | + | We did a PCR amplification for BphR1, BphR2 and BphA1 genes from strain ''Pseudomonas pseudoalcaligenes''. Products were good and purified following the protocol. |
| - | + | ||
| - | + | ||
| - | + | Protocol : [http://www.mn-net.com/tabid/1452/default.aspx Gel purification ] | |
| - | + | ||
| - | + | ||
===='''2 - Digestion PCR products : BphR2, BphR1, BphA1 and digestion of pSB1C3'''==== | ===='''2 - Digestion PCR products : BphR2, BphR1, BphA1 and digestion of pSB1C3'''==== | ||
| Line 50: | Line 46: | ||
** H2O : 10.5µL | ** H2O : 10.5µL | ||
| - | * | + | * pSB1C3 : |
| - | ** | + | ** pSB1C3 : 4µL |
** Buffer orange : 0.8µL | ** Buffer orange : 0.8µL | ||
** EcoRI : 0.5µL | ** EcoRI : 0.5µL | ||
| Line 59: | Line 55: | ||
We let our digestion 1h30 at 37°C. | We let our digestion 1h30 at 37°C. | ||
| - | ===='''3 - | + | ===='''3 - Inactivation of EcoRI/PstI used for the digestion of PCR products : BphR1, BphR2, BphA1 and pSB1C3'''==== |
Anaïs, Sheng | Anaïs, Sheng | ||
| Line 69: | Line 65: | ||
* BphR1 : 97.9ng/µL | * BphR1 : 97.9ng/µL | ||
* BphR2 : 24.2ng/µL | * BphR2 : 24.2ng/µL | ||
| - | * | + | * pSB1C3 : 36.1ng/µL |
| - | ===='''4 - Ligation of BphA1, BphR1, BphR2 and | + | ===='''4 - Ligation of BphA1, BphR1, BphR2 and pSB1C3'''==== |
Zhou | Zhou | ||
Used quantities : | Used quantities : | ||
| - | * | + | * pSB1C3 : 2µL |
* DNA : 2µL | * DNA : 2µL | ||
* Buffer ligase : 2µL | * Buffer ligase : 2µL | ||
Latest revision as of 23:56, 4 October 2013
Notebook : July 9
Lab work
Objective : obtaining biobricks in pSB3K3
1 - Transformation of BBa_J04450 in DH5α
Anaïs
Protocol : Bacterial transformation
B - PCB sensor system
Objective : obtaining BBa_K1155001, BBa_K1155002 and BphR2 protein
1 - PCR purification of BphR1, BphR2 and BphA1
Zhou
We did a PCR amplification for BphR1, BphR2 and BphA1 genes from strain Pseudomonas pseudoalcaligenes. Products were good and purified following the protocol.
Protocol : [http://www.mn-net.com/tabid/1452/default.aspx Gel purification ]
2 - Digestion PCR products : BphR2, BphR1, BphA1 and digestion of pSB1C3
Abdou, Anaïs
Used quantities :
- BphA1 :
- DNA : 8µL
- Buffer FD : 1.6µL
- EcoRI FD : 1µL
- PstI FD : 1µL
- H2O : 4.4µL
- BphR1, BphR2 :
- DNA : 15µL
- Buffer FD : 3µL
- EcoRI FD : 0.75µL
- PstI FD : 0.75µL
- H2O : 10.5µL
- pSB1C3 :
- pSB1C3 : 4µL
- Buffer orange : 0.8µL
- EcoRI : 0.5µL
- PstI : 0.5µL
- H2O : 2.2µL
We let our digestion 1h30 at 37°C.
3 - Inactivation of EcoRI/PstI used for the digestion of PCR products : BphR1, BphR2, BphA1 and pSB1C3
Anaïs, Sheng
Protocol : Ethanol precipitation
Nanodrop :
- BphA1 : 108.3ng/µL
- BphR1 : 97.9ng/µL
- BphR2 : 24.2ng/µL
- pSB1C3 : 36.1ng/µL
4 - Ligation of BphA1, BphR1, BphR2 and pSB1C3
Zhou
Used quantities :
- pSB1C3 : 2µL
- DNA : 2µL
- Buffer ligase : 2µL
- Ligase : 1µL
- H2O : 13µL
Human Practices
We made the thrid meeting about open source : Open Source Reflexion
| Previous day | Back to calendar | Next day |
