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Team:Paris Saclay/Notebook/July/26
From 2013.igem.org
(Difference between revisions)
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==='''A - Aerobic/Anaerobic regulation system'''=== | ==='''A - Aerobic/Anaerobic regulation system'''=== | ||
| - | ===='''Objective : obtaining | + | ===='''Objective : obtaining BBa_K1155003'''==== |
===='''1 - Denaturation of EcoRI/SpeI used for the digestion of PCR products : RBS-AmilCP '''==== | ===='''1 - Denaturation of EcoRI/SpeI used for the digestion of PCR products : RBS-AmilCP '''==== | ||
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We used 90µL of DNA. | We used 90µL of DNA. | ||
| - | + | At the end, we mix our DNA 20µL or water. | |
===='''2 - Ligation of RBS-AmilCP and Term-PSB1C3'''==== | ===='''2 - Ligation of RBS-AmilCP and Term-PSB1C3'''==== | ||
| Line 30: | Line 30: | ||
* H2O : 3.5µL | * H2O : 3.5µL | ||
| - | ===='''Objective : obtaining | + | ===='''Objective : obtaining BBa_K1155004, BBa_K1155005, BBa_K1155006'''==== |
===='''1 - Electrophoresis of PCR products : NarK, NarG, NirB'''==== | ===='''1 - Electrophoresis of PCR products : NarK, NarG, NirB'''==== | ||
| Line 142: | Line 142: | ||
==='''B - PCB sensor system'''=== | ==='''B - PCB sensor system'''=== | ||
| - | ===='''Objective : obtaining | + | ===='''Objective : obtaining BBa_K1155002'''==== |
| - | ===='''1 - Tranformation of | + | ===='''1 - Tranformation of BBa_K1155002 in DH5α '''==== |
Abdou, Xavier | Abdou, Xavier | ||
Revision as of 09:36, 30 September 2013
Notebook : July 26
Lab work
A - Aerobic/Anaerobic regulation system
Objective : obtaining BBa_K1155003
1 - Denaturation of EcoRI/SpeI used for the digestion of PCR products : RBS-AmilCP
Abdou
Protocol : Ethanol precipitation
We used 90µL of DNA. At the end, we mix our DNA 20µL or water.
2 - Ligation of RBS-AmilCP and Term-PSB1C3
Xavier
Used quantities :
- RBS-AmilCP : 2µL
- Term in PSB1C3 : 2.5µL
- Buffer : 1µL
- Ligase : 1µL
- H2O : 3.5µL
Objective : obtaining BBa_K1155004, BBa_K1155005, BBa_K1155006
1 - Electrophoresis of PCR products : NarK, NarG, NirB
Abdou
| [[]] |
|
Expected sizes :
- NarK, NarG, NirB : ...
|
We lost all our PCR products. We will do PCR again using more concentrated DNA and oligos. |
2 - PCR of NarK, NarG, NirB
Abdou, Xavier
Used quantities :
- NarK :
- Buffer phusion : 10µL
- dNTP : 1µL
- Oligo ... : 2µL
- Oligo ... : 2µL
- Concentrated DNA : 2µL
- Phusion : 0.25µL
- NarG :
- Buffer phusion : 10µL
- dNTP : 1µL
- Oligo ... : 2µL
- Oligo ... : 2µL
- Concentrated DNA : 2µL
- Phusion : 0.25µL
- NirB :
- Buffer phusion : 10µL
- dNTP : 1µL
- Oligo ... : 2µL
- Oligo ... : 2µL
- Concentrated DNA : 2µL
- Phusion : 0.25µL
PCR Program :
3 - Electrophoresis of PCR products : NarK, NarG, NirB
Abdou, Xavier
| [[]] |
|
Expected sizes :
- NarK, NarG, NirB : ...
|
We obtain fragments at the right size. We can purify it. |
4 - Gel purification of PCR products : NarK, NarG, NirB
Xavier
Protocol : [http://www.mn-net.com/tabid/1452/default.aspx Gel purification ]
REPRIS dans 20µL !!!!!!!!
5 - Digestion of PCR products : NarK, NarG, NirB by EcoRI/PstI
Abdou
Quantities used :
- NarK, NarG, NirB : 10µL
- Buffer FD : 3µL
- EcoRI FD : 1.5µL
- PstI FD : 1.5µL
- H2O : 14µL
We let the digestion at 37°C during 1h30 and then at -20°C.
B - PCB sensor system
Objective : obtaining BBa_K1155002
1 - Tranformation of BBa_K1155002 in DH5α
Abdou, Xavier
Protocol : Bacterial tranformation
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