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Team:Paris Saclay/Notebook/August/26
From 2013.igem.org
(Difference between revisions)
| Line 7: | Line 7: | ||
==='''A - Aerobic/Anaerobic regulation system'''=== | ==='''A - Aerobic/Anaerobic regulation system'''=== | ||
| - | ===='''Objective : characterize | + | ===='''Objective : characterize BBa_K1155000, BBa_K1155004, BBa_K1155005, BBa_K1155006'''==== |
| - | ===='''1 - Digestion of | + | ===='''1 - Digestion of BBa_K1155000, BBa_K1155004, BBa_K1155005, BBa_K1155006 by SpeI/PstI'''==== |
XiaoJing | XiaoJing | ||
| - | * | + | * BBa_K1155000 : |
** DNA : 14µL | ** DNA : 14µL | ||
** SpeI FD : 2µL | ** SpeI FD : 2µL | ||
| Line 19: | Line 19: | ||
** Buffer FD : 2µL | ** Buffer FD : 2µL | ||
| - | * | + | * BBa_K1155004 clone 6 : |
** DNA : 14µL | ** DNA : 14µL | ||
** SpeI FD : 2µL | ** SpeI FD : 2µL | ||
| Line 25: | Line 25: | ||
** Buffer FD : 2µL | ** Buffer FD : 2µL | ||
| - | * | + | * BBa_K1155004 clone 7 : |
** DNA : 14µL | ** DNA : 14µL | ||
** SpeI FD : 2µL | ** SpeI FD : 2µL | ||
| Line 31: | Line 31: | ||
** Buffer FD : 2µL | ** Buffer FD : 2µL | ||
| - | * | + | * BBa_K1155005 clone 6 : |
** DNA : 7µL | ** DNA : 7µL | ||
** SpeI FD : 2µL | ** SpeI FD : 2µL | ||
| Line 38: | Line 38: | ||
** H2O : 7µL | ** H2O : 7µL | ||
| - | * | + | * BBa_K1155005 clone 7 : |
** DNA : 14µL | ** DNA : 14µL | ||
** SpeI FD : 2µL | ** SpeI FD : 2µL | ||
| Line 44: | Line 44: | ||
** Buffer FD : 2µL | ** Buffer FD : 2µL | ||
| - | * | + | * BBa_K1155006 clone 6 : |
** DNA : 4µL | ** DNA : 4µL | ||
** SpeI FD : 2µL | ** SpeI FD : 2µL | ||
| Line 51: | Line 51: | ||
** H2O : 10µL | ** H2O : 10µL | ||
| - | + | * BBa_K1155004 already digested by SpeI : | |
** DNA : 13µL | ** DNA : 13µL | ||
** PstI FD : 2µL | ** PstI FD : 2µL | ||
| Line 57: | Line 57: | ||
** H2O : 3µL | ** H2O : 3µL | ||
| - | + | * BBa_K1155005 already digested by SpeI : | |
** DNA : 10µL | ** DNA : 10µL | ||
** PstI FD : 2µL | ** PstI FD : 2µL | ||
| Line 63: | Line 63: | ||
** H2O : 6µL | ** H2O : 6µL | ||
| - | * | + | * BBa_K1155006 already digested by SpeI : |
** DNA : 9µL | ** DNA : 9µL | ||
** PstI FD : 2µL | ** PstI FD : 2µL | ||
| Line 71: | Line 71: | ||
We keep digestions for 30 minutes at 37°C. | We keep digestions for 30 minutes at 37°C. | ||
| - | ===='''2 - Denaturation of SpeI/PstI used for the digestion of | + | ===='''2 - Denaturation of SpeI/PstI used for the digestion of BBa_K1155000, BBa_K1155004, BBa_K1155005, BBa_K1155006'''==== |
XiaoJing | XiaoJing | ||
Revision as of 08:58, 30 September 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 keep digestions for 30 minutes at 37°C.
2 - Denaturation 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 :
- Pfnr : 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 keep 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
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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