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Team:Paris Saclay/Notebook/August/26
From 2013.igem.org
(→4 - Transformation of ligation of Pfnr and NirB with RBS-LacZ-Term or RBS-Amil CP-Term in PSB1C3) |
(→2 - Inactivation of SpeI/PstI used for the digestion of BBa_K1155000, BBa_K1155004, BBa_K1155005, BBa_K1155006) |
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| Line 80: | Line 80: | ||
Nanodrop : | Nanodrop : | ||
| - | * | + | * Pndh* : 91.8ng/µL |
* NarK clone 6 : 19.8ng/µL | * NarK clone 6 : 19.8ng/µL | ||
* Nark already digested by SpeI : 15.1ng/µL | * Nark already digested by SpeI : 15.1ng/µL | ||
Revision as of 22:15, 4 October 2013
Notebook : August 26
Lab work
A - Aerobic/Anaerobic regulation system
Objective : characterize BBa_K1155000, BBa_K1155004, BBa_K1155005, BBa_K1155006
1 - Digestion of BBa_K1155000, BBa_K1155004, BBa_K1155005, BBa_K1155006 by SpeI/PstI
XiaoJing
- BBa_K1155000 :
- DNA : 14µL
- SpeI FD : 2µL
- PstI FD : 2µL
- Buffer FD : 2µL
- BBa_K1155004 clone 6 :
- DNA : 14µL
- SpeI FD : 2µL
- PstI FD : 2µL
- Buffer FD : 2µL
- BBa_K1155004 clone 7 :
- DNA : 14µL
- SpeI FD : 2µL
- PstI FD : 2µL
- Buffer FD : 2µL
- BBa_K1155005 clone 6 :
- DNA : 7µL
- SpeI FD : 2µL
- PstI FD : 2µL
- Buffer FD : 2µL
- H2O : 7µL
- BBa_K1155005 clone 7 :
- DNA : 14µL
- SpeI FD : 2µL
- PstI FD : 2µL
- Buffer FD : 2µL
- BBa_K1155006 clone 6 :
- DNA : 4µL
- SpeI FD : 2µL
- PstI FD : 2µL
- Buffer FD : 2µL
- H2O : 10µL
- BBa_K1155004 already digested by SpeI :
- DNA : 13µL
- PstI FD : 2µL
- Buffer FD : 2µL
- H2O : 3µL
- BBa_K1155005 already digested by SpeI :
- DNA : 10µL
- PstI FD : 2µL
- Buffer FD : 2µL
- H2O : 6µL
- BBa_K1155006 already digested by SpeI :
- DNA : 9µL
- PstI FD : 2µL
- Buffer FD : 2µL
- H2O : 7µL
We incubate the digestion for 30 minutes at 37°C.
2 - Inactivation of SpeI/PstI used for the digestion of BBa_K1155000, BBa_K1155004, BBa_K1155005, BBa_K1155006
XiaoJing
Protocol : Ethanol precipitation
We used 20µL of DNA.
Nanodrop :
- Pndh* : 91.8ng/µL
- NarK clone 6 : 19.8ng/µL
- Nark already digested by SpeI : 15.1ng/µL
- NarG clone 6 : 13.4ng/µL
- NarG clone 7 : 103.9ng/µL
- narG already digested by SpeI : 17.4ng/µL
- NirB clone 6 : 46ng/µL
- NirB clone 7 : 61.6ng/µL
- NirB already digested by SpeI : 16.1ng/µL
|
According to the result of the Nanodrop, we decide to do ligations with Pfnr and nirB clone 6. |
3 - Ligation of Pfnr, NirB clone 6 with RBS-LacZ-Term or RBS-Amil CP-Term in pSB1C3
XiaoJing
Used quantities :
NirB clone 6 with RBS-LacZ or RBS-Amil CP, Pfnr with RBS-LacZ or RBS-Amil CP :
- RBS-LacZ-Term or RBS-Amil CP-Term : 3µL
- Pfnr or NirB clone 6 : 5µL
- Buffer ligase : 2µL
- Ligase T4 : 2µL
- H20 : 8µL
We incubate the ligation for 1h at 37°C.
4 - Transformation of ligation of Pfnr and NirB with RBS-LacZ-Term or RBS-Amil CP-Term in PSB1C3
XiaoJing
Protocol : Bacterial transformation
5- Purification colony from strain MG1655Z1 Δfnr::Km containing plasmid pcp20
XioaJing
|
Transformation of 08/19/13 works. We will purify our colonies. |
We streaked colonies into ampicilin and chloramphenicol plates and incubate at 30°C.
A - Aerobic/Anaerobic regulation system / B - PCB sensor system
Objective : obtaining FNR and BphR2
1 - Gibson assembly
XiaoJing
|
Tranformation of 08/21 didn't work. We will do the Gibson assembly again. |
Used quantities :
- RBS-BphR2 :
- PSB1C3 : 3µL
- BphR2 Part I : 1µL
- BphR2 Part II : 1µL
- Gibson mix : 15µL
- FNR :
- PSB1C3 : 3µL
- FNR Part I : 1µL
- FNR Part II : 1µL
- Gisbon mix : 15µL
- RBS-FNR :
- PSB1C3 : 3µL
- RBS-FNR Part I : 1µL
- FNR Part II : 1µL
- Gibson mix : 15µL
We keep these mix at 50°C for 1h.
2 - Transformation of FNR, RBS-FNR and RBS-BphR2 in DH5α
XiaoJing
Protocol : Bacterial transformation
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