Team:Paris Saclay/Notebook/August/21
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==='''A - Aerobic/Anaerobic regulation system / B - PCB sensing system'''=== | ==='''A - Aerobic/Anaerobic regulation system / B - PCB sensing system'''=== | ||
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Xiaojing | Xiaojing | ||
Revision as of 21:04, 8 September 2013
Contents |
Notebook : August 21
Lab work
A - Aerobic/Anaerobic regulation system
Obtaining FNR Promoter (nirB,narG,narK)plus RBS_LacZ+Term_PSB1C3 and FNR Promoter (nirB,narG,narK)plus RBS_Amicip+Term_PSB1C3
1 - Colony PCR on e.coli with FNR Promoter (nirB,narG,narK)plus RBS_LacZ+Term_PSB1C3 and FNR Promoter (nirB,narG,narK)plus RBS_Amicip+Term_PSB1C3 for 8 colonies of each
Damir,Xiaojing
- A1-8: FNR Promoter nirB plus RBS_LacZ+Term_PSB1C3
- B1-8: FNR Promoter nirB plus RBS_Amicip+Term_PSB1C3
- C1-8: FNR Promoter narG plus RBS_LacZ+Term_PSB1C3
- D1-8: FNR Promoter narG plus RBS_Amicip+Term_PSB1C3
- E1-8: FNR Promoter narK plus RBS_LacZ+Term_PSB1C3
- F1-8: FNR Promoter narK Plus RBS_Amicip+Term_PSB1C3
- Picking of 48 colonies
- Preparation Master mix A
- H2O : 1880µL
- dNTP : 50µL
- VR primer : 50µL
- DreamTaq buffer 10x : 250µL
- DreamTaq enzyme : 20µL
- Preparation Master mix B
- Master mix A :1125µL
- VF2 primer : 25µL
Protocol : Colony PCR By using Master mix B
PCR Program :
2 - Gel electrophoresis of the colony PCR products
Damir
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Expected size : 3583bp
A - Aerobic/Anaerobic regulation system / B - PCB sensing system
1 - Gibson assembly.
Xiaojing
- RBS_BphR2_part1, BphR2_part2 and plasmid PSB1C3
- FNR_part1, FNR part1 and plasmid PSB1C3
- RBS_FNR part1, FNR_part2 and plasmid PSB1C3
Sample Volume:
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These 3 mixture is incubated at 50°C for up to one hour. Transformation and spread in 6 LB plates with Chlorenphenicol . Incubate at 37°C over night.
3 - PCR pbaroduct (made the 08/01/2013) purification
Damirr available quantity:
- FNR Part1 : 10 µl
- FNR Part2 : 19 µl
- RBS FNR Part1 :16.1µl
- RBS BphR2 Part1 : 28µl
- BphR2 Part1 : 16.4 µl
- BphR2 Part2 : 18.9 µl
Protocol : kit purification
Manipulation error : The elution step was made using the recuperation tube from the filtering step, instead of a new, clean eppendorf tube.