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Team:Lyon-INSA-ENS/Realisation/Week5
From 2011.igem.org
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| - | Additional minipreps of the same 6 parts | + | Additional minipreps using the QuickPure protocol of the same 6 parts ( 5 replica each ) <br/> |
| + | Digestion as previously<br/><br/> | ||
| + | |||
| + | Ligation to obtain : RBS ( strong and weak ) + GFP, RBS ( strong and weak ) + YFP. This corresponds respectively to 2M+2L, 5L+2L, 2M+24E, 5L+24E assemblies.<br/><br/> | ||
| + | |||
| + | Start of a 5mL culture of NM522 cells.<br/> | ||
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<p style = "line-height : 1.5em"> | <p style = "line-height : 1.5em"> | ||
| - | Transformation of the ligations into NM522 using a CaCl2 chemical transformation. | + | Start of a 50mL culture of NM522 from the overnight preculture ( 50mL sterile LB, 250µL of preculture ).<br/> |
| + | |||
| + | Transformation of 5µL ( S series ) or 10 µL ( D series ) from the previous ligations into NM522 (V=15mL) using a CaCl2 chemical transformation. Positive control was done with 1µL Puc18, negative with 5µL water. <br/> | ||
</p> | </p> | ||
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<br/> | <br/> | ||
<p style = "line-height : 1.5em"> | <p style = "line-height : 1.5em"> | ||
| - | + | Miniprep using the QuickPure protocol of the previous liquid cultures.<br/><br/> | |
| + | |||
| + | Digestion of the plasmids by X+P.<br/><br/> | ||
| + | |||
| + | Electrophoresis of the digested and non digested plasmids : <br/> | ||
| + | 2M+2L S1 : no DNA <br/> | ||
| + | 5L+2L S1 : 800 bp insert <br/> | ||
| + | 2M+2L S2 : no DNA <br/> | ||
| + | 2M+2L D1 : 800 bp insert <br/> | ||
| + | 5L+24E S2 : no insert <br/> | ||
| + | 5L+2L D2 : 800 bp insert <br/> | ||
| + | 2M+24E S2 : no insert <br/> | ||
| + | 2M+24E D2 : 800 bp insert <br/><br/> | ||
| + | |||
| + | However, due to the small size of the RBS, a flaw in our protocol ( choice of antibiotic resistances ) doesn't allow us to detect a difference between 24E or 2L plasmids compared to their ligated equivalents. Thus we can't conclude that the 800 bp inserts correspond to the ligated parts. | ||
| + | |||
</p> | </p> | ||
Revision as of 08:08, 27 July 2011
Week 5
From Monday the 4th of July to Friday the 8th of July 2011
Monday
Additional minipreps using the QuickPure protocol of the same 6 parts ( 5 replica each )
Digestion as previously
Ligation to obtain : RBS ( strong and weak ) + GFP, RBS ( strong and weak ) + YFP. This corresponds respectively to 2M+2L, 5L+2L, 2M+24E, 5L+24E assemblies.
Start of a 5mL culture of NM522 cells.
Tuesday
Start of a 50mL culture of NM522 from the overnight preculture ( 50mL sterile LB, 250µL of preculture ).
Transformation of 5µL ( S series ) or 10 µL ( D series ) from the previous ligations into NM522 (V=15mL) using a CaCl2 chemical transformation. Positive control was done with 1µL Puc18, negative with 5µL water.
Wednesday
Selection of individual transformed colonies and start of solid and liquid culture.
We shoot our "Cobalt Buster's Clip" which introduces, in a funny way, our team : students, instructors and advisors. We dedicate the all afternoon to this shooting.
Thursday
Miniprep using the QuickPure protocol of the previous liquid cultures.
Digestion of the plasmids by X+P.
Electrophoresis of the digested and non digested plasmids :
2M+2L S1 : no DNA
5L+2L S1 : 800 bp insert
2M+2L S2 : no DNA
2M+2L D1 : 800 bp insert
5L+24E S2 : no insert
5L+2L D2 : 800 bp insert
2M+24E S2 : no insert
2M+24E D2 : 800 bp insert
However, due to the small size of the RBS, a flaw in our protocol ( choice of antibiotic resistances ) doesn't allow us to detect a difference between 24E or 2L plasmids compared to their ligated equivalents. Thus we can't conclude that the 800 bp inserts correspond to the ligated parts.
