"
Team:Lyon-INSA-ENS/Realisation/Week9
From 2011.igem.org
| Line 79: | Line 79: | ||
Transformation and plating of S19 on LB+Amp with : p157(3µL), p115(3µL), 3 replica each.<br/><br/> | Transformation and plating of S19 on LB+Amp with : p157(3µL), p115(3µL), 3 replica each.<br/><br/> | ||
| - | + | . | |
| - | Extraction of pIG16 from S19 | + | Extraction of pIG16 from S19 with the QIAGen miniprep protocol |
</p> | </p> | ||
| Line 93: | Line 93: | ||
<p style=" line-height : 1.5em"> | <p style=" line-height : 1.5em"> | ||
| - | The transformation gave too many bacteria : plating of all the previous transformed strains on a new Petri dish.<br/><br/> | + | The transformation gave too many bacteria : plating of all the previous transformed strains on a new Petri dish in order to isolate individual clones.<br/><br/> |
Start of 5mL cultures for 24 well plates :<br/> | Start of 5mL cultures for 24 well plates :<br/> | ||
M63 : PHL818, NM522/pUC18 (Amp), NM522/pSB1C3(Cm)<br/> | M63 : PHL818, NM522/pUC18 (Amp), NM522/pSB1C3(Cm)<br/> | ||
LB/2 : PHL818, NM522/pUC18 (Amp), NM522/pSB1C3(Cm), S17 (Cm), S18(Amp), NM522<br/> | LB/2 : PHL818, NM522/pUC18 (Amp), NM522/pSB1C3(Cm), S17 (Cm), S18(Amp), NM522<br/> | ||
| - | LB : NM522/pIG16/p157, NM522/pIG16/p115, NM522/pIG16/p127, NM522/pIG16/p116<br/><br/> | + | LB : NM522/pIG16/p157, NM522/pIG16/p115, NM522/pIG16/p127, NM522/pIG16/p116, NM522<br/><br/> |
Digestion of pIG16 (E+P) and electrophoresis : the digestion had failed or the extracted plasmid was not pIG16.<br/> | Digestion of pIG16 (E+P) and electrophoresis : the digestion had failed or the extracted plasmid was not pIG16.<br/> | ||
Digestion of pIG16 by E,X,S,P,E+P,X+S : E,X and S show a linearization of the plasmid, P shows nothing, E+P shows a linearization and X+S extracts the part -> P was inactive. <br/><br/> | Digestion of pIG16 by E,X,S,P,E+P,X+S : E,X and S show a linearization of the plasmid, P shows nothing, E+P shows a linearization and X+S extracts the part -> P was inactive. <br/><br/> | ||
| - | |||
</p> | </p> | ||
Revision as of 13:08, 19 August 2011
Week 9
From Monday the 15th of August to Friday the 19th of August 2011
Monday
Tuesday
Start of 5mL cultures for adherence tests from the collection :
LB : NM522
LB/2 : NM522, S3, S4, S15, S19
M63 : NM522, S15, S3, S19, S4, S18
Plating of individual or non individual clones from the previous NM522-pIG16 and NM522-pIG3 cultures on LB+amp medium. Incubation at 37°C overnight.
Wednesday
Start of 3x5mL cultures of NM522 in LB medium from 50µL of a saturated NM522 culture for a later transformation.
Start of 5mL cultures of the individual clones plated on the previous day.
Test of antibiotics : start of 5mL cultures ( 5mL LB, 50µL antibiotic, 10µL saturated NM522) of NM522 with antibiotics to test if the antibiotic is still good for Kan, Tet, Cm and Amp
The bacteria with Amp grow : a new solution is made ( 5x1mL mother solutions at 100mg/mL from solid Amp, and 2x10mL solutions at 100µg/mL by dilution of 10µL of mother solution into 10mL sterile water )
Start of 24 well plate adherence test in M63G medium with PHL818 ( positive control ), NM522 ( negative control ), S4 + Amp + CoCl2 ( in increasing concentration )
Transformation and plating of NM522 on LB+Amp with : pIG6 (3µL), pIG7 (3µL), pIG24 (2µL), p56 (2µL), p10 (2µL), pIG16+p157 (3µL each), pIG16+p115(3µL each), pIG16+p127(3µL each), pIG16+p116 ( 3µL and 5µL respectively ), pIG25 (1µL).
Transformation and plating of S19 on LB+Amp with : p157(3µL), p115(3µL), 3 replica each.
.
Extraction of pIG16 from S19 with the QIAGen miniprep protocol
Thursday
The transformation gave too many bacteria : plating of all the previous transformed strains on a new Petri dish in order to isolate individual clones.
Start of 5mL cultures for 24 well plates :
M63 : PHL818, NM522/pUC18 (Amp), NM522/pSB1C3(Cm)
LB/2 : PHL818, NM522/pUC18 (Amp), NM522/pSB1C3(Cm), S17 (Cm), S18(Amp), NM522
LB : NM522/pIG16/p157, NM522/pIG16/p115, NM522/pIG16/p127, NM522/pIG16/p116, NM522
Digestion of pIG16 (E+P) and electrophoresis : the digestion had failed or the extracted plasmid was not pIG16.
Digestion of pIG16 by E,X,S,P,E+P,X+S : E,X and S show a linearization of the plasmid, P shows nothing, E+P shows a linearization and X+S extracts the part -> P was inactive.
Friday
