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Team:Lyon-INSA-ENS/Realisation/Week7
From 2011.igem.org
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<div class="cadre" ; style="background-color:green;" > | <div class="cadre" ; style="background-color:green;" > | ||
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| - | <h1 style="color: white;"> Week 7 </h1> | + | <h1 style="color: white;"> Week 7 </h1> |
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| - | <h6 style="text-align :left"> Monday </h6> | + | <h6 style="text-align :left"> Monday </h6> <HR> |
| - | + | <br/> | |
<p style=" line-height : 1.5em"> | <p style=" line-height : 1.5em"> | ||
| - | Concerned parts : RBS-GFP-LVA (Cn),BBa_J23119 ( 18A, plate 1 )(Amp), Curli (Cn), ompR234 (Cn) <br/><br/> | + | Concerned parts : RBS-GFP-LVA (Cn),BBa_J23119 ( 18A, plate 1 )(Amp), Curli (Cn), ompR234 (Cn) |
| - | + | <br/><br/> | |
Start of 5mL liquid cultures from a Petri dish culture: <br/> | Start of 5mL liquid cultures from a Petri dish culture: <br/> | ||
| - | 5mL sterile LB <br/> | + | 5mL sterile LB |
| - | 50µL antibiotic <br/> | + | <br/> |
| - | bacteria <br/> | + | 50µL antibiotic |
| - | Incubation at 37°C <br/><br/> | + | <br/> |
| - | + | bacteria | |
| - | Later, start of 150mL liquid cultures from the previous 5mL cultures <br/> | + | <br/> |
| - | 150 mL sterile LB <br/> | + | Incubation at 37°C |
| - | 300 µL from the grown 5mL bacterial culture <br/> | + | <br/><br/> |
| - | 1.5 mL antibiotic ( Amp or Cn ) <br/> | + | Later, start of 150mL liquid cultures from the previous 5mL cultures |
| - | Incubation at 37°C overnight. <br/><br/><br/> | + | <br/> |
| - | + | 150 mL sterile LB | |
| + | <br/> | ||
| + | 300 µL from the grown 5mL bacterial culture | ||
| + | <br/> | ||
| + | 1.5 mL antibiotic ( Amp or Cn ) | ||
| + | <br/> | ||
| + | Incubation at 37°C overnight. | ||
| + | <br/><br/><br/> | ||
10X concentration and plating of 100 µL of transformed bacteria from the previous week : 2M+24E ( amp ), Curli + GFP ( Kan ), Kan + 2L ( Kan ), positive control ( Puc18 plasmid ), negative control ( water ) <br/> | 10X concentration and plating of 100 µL of transformed bacteria from the previous week : 2M+24E ( amp ), Curli + GFP ( Kan ), Kan + 2L ( Kan ), positive control ( Puc18 plasmid ), negative control ( water ) <br/> | ||
Incubation at 37°C <br/> | Incubation at 37°C <br/> | ||
| - | |||
</p> | </p> | ||
<br/> <br/> | <br/> <br/> | ||
| - | <h6 style="text-align :left"> Tuesday </h6> | + | <h6 style="text-align :left"> Tuesday </h6> <HR> |
<br/> <br/> | <br/> <br/> | ||
Revision as of 14:42, 26 July 2011
Week 7
From Monday the 18th of July to Friday the 22nd of July 2011
Monday
Concerned parts : RBS-GFP-LVA (Cn),BBa_J23119 ( 18A, plate 1 )(Amp), Curli (Cn), ompR234 (Cn)
Start of 5mL liquid cultures from a Petri dish culture:
5mL sterile LB
50µL antibiotic
bacteria
Incubation at 37°C
Later, start of 150mL liquid cultures from the previous 5mL cultures
150 mL sterile LB
300 µL from the grown 5mL bacterial culture
1.5 mL antibiotic ( Amp or Cn )
Incubation at 37°C overnight.
10X concentration and plating of 100 µL of transformed bacteria from the previous week : 2M+24E ( amp ), Curli + GFP ( Kan ), Kan + 2L ( Kan ), positive control ( Puc18 plasmid ), negative control ( water )
Incubation at 37°C
Tuesday
Midiprep to extract DNA from the previous liquid cultures.
Nanodrop quantification :
OmpR234 : 59.5 ng/µL
GFP : 109.5 ng/µL
18A : 73.7 ng/µL
Curli : 82.6 ng/µL
The 260/280 ratio is lower than 2 in all cases.
Wednesday
Digestion of several biobricks : RBS, GFP, YFP, Curli, OmpR234. Electrophoresis to control the digestions.
Thursday
Standard or 3A ligation of the previously cut biobricks.TSS transformation into NM522.
Friday
Start of 5mL liquid cultures of selected individual colonies. Extraction of the plasmids to test the presence of the insert.
