From Monday the 15th of August to Friday the 19th of August 2011

Monday

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Digestion with E+X, ligation with ...
Transformation and plating of NM522 with : pIG3 (3µL), pIG16 (3µL)

Tuesday

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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

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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 ), S18 + 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

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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

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Plating of NM522/pIG16/p157, NM522/pIG16/p115, NM522/pIG16/p127, NM522/pIG16/p116 ( who have a double resistance ) on their second resistance ( Kan ).
Extraction (QIAgen) and digestion(Fermentas) of these plasmids by E : only one plasmid was in the strains -> the transformations need to be restarted.

Start of 24 well plates :
M63 : PHL818 ( positive control ), NM522 and NM522/pUC18( negative controls ), S18 + Co ( in increasing concentration).
M63 : PHL818, NM522, NM522/pUC18 (with and without cobalt), S17 and S18 ( with cobalt in increasing concentrations )
LB/2 : PHL818, NM522, NM522/pUC18 (with and without cobalt), S17 and S18 ( with cobalt in increasing concentrations )

The NM522/pIG24 (Cm) strain grows on Amp plates and not Cm. Moreover, pIG24 is a linear plasmid and should not have transformed : plating of NM522, NM522/pIG24 on LB+Amp and start of 5mL cultures ( LB+Amp ) of the same strains to test Amp efficiency or contamination of NM522 with an AmpR strain.