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Team:Lyon-INSA-ENS/Realisation/Week9
From 2011.igem.org
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<p style=" line-height : 1.5em"> | <p style=" line-height : 1.5em"> | ||
| - | + | <FONT COLOR="red"> <b>Transformations and controls for future tests</b> </FONT> <br/><br/> | |
<b>Digestion</b> with E+X, <b>ligation</b> with ... <br> | <b>Digestion</b> with E+X, <b>ligation</b> with ... <br> | ||
<b>Transformation and plating</b> of NM522 with : pIG3 (3µL), pIG16 (3µL)<br/> | <b>Transformation and plating</b> of NM522 with : pIG3 (3µL), pIG16 (3µL)<br/> | ||
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<p style=" line-height : 1.5em"> | <p style=" line-height : 1.5em"> | ||
| + | <FONT COLOR="red"> <b>Transformations and controls for future tests</b> </FONT> <br/><br/> | ||
| + | Plating of individual or non individual clones from the previous NM522-pIG16 and NM522-pIG3 cultures on LB+amp medium.<br/> Incubation at 37°C overnight.<br/><br/><br/> | ||
| + | </p> | ||
| + | |||
| + | <p style=" line-height : 1.5em"> | ||
| + | <FONT COLOR="purple"> <b>Adherence Test</b> </FONT> <br/><br/> | ||
Start of 5mL cultures for adherence tests from the collection : <br/> | Start of 5mL cultures for adherence tests from the collection : <br/> | ||
LB : NM522 <br/> | LB : NM522 <br/> | ||
LB/2 : NM522, S3, S4, S15, S19 <br/> | LB/2 : NM522, S3, S4, S15, S19 <br/> | ||
| - | M63 : NM522, S15, S3, S19, S4, S18 < | + | M63 : NM522, S15, S3, S19, S4, S18 |
| + | </p> | ||
| + | |||
| - | |||
</p> | </p> | ||
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<br/> | <br/> | ||
| - | + | <p style=" line-height : 1.5em"> | |
| + | <FONT COLOR="red"> <b>Transformations and controls for future tests</b> </FONT> <br/><br/> | ||
Start of 3x5mL cultures of NM522 in LB medium from 50µL of a saturated NM522 culture for a later transformation.<br/> | Start of 3x5mL cultures of NM522 in LB medium from 50µL of a saturated NM522 culture for a later transformation.<br/> | ||
Start of 5mL cultures of the individual clones plated on the previous day. <br/><br/> | Start of 5mL cultures of the individual clones plated on the previous day. <br/><br/> | ||
| + | <b>Transformation</b> and <b>plating</b> 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). <br/><br/> | ||
| + | |||
| + | <b>Transformation</b> and <b>plating</b> of S19 on LB+Amp with : p157(3µL), p115(3µL), 3 replica each.<br/><br/> | ||
| + | |||
| + | <b>Extraction</b> of pIG16 from S19 with the QIAGen miniprep protocol. <br/><br/><br/> | ||
| + | |||
| + | </p> | ||
| + | |||
| + | |||
| + | <p style=" line-height : 1.5em"> | ||
| + | <FONT COLOR="purple"> <b>Adherence Test</b> </FONT> <br/><br/> | ||
| + | Start of <b>24 well plate</b> adherence test in M63G medium with PHL818 ( positive control ), NM522 ( negative control ), S18 + Amp + CoCl2 ( in increasing concentration ) <br/> <br/><br/> | ||
| + | |||
| + | </p> | ||
| + | |||
| + | <p style=" line-height : 1.5em"> | ||
| + | <FONT COLOR="orange"> <b>Others</b> </FONT> <br/><br/> | ||
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<br/> | 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<br/> | ||
| - | 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 )< | + | 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 ). |
| + | </p> | ||
| + | |||
| - | |||
| - | |||
| - | |||
| - | |||
</p> | </p> | ||
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<p style=" line-height : 1.5em"> | <p style=" line-height : 1.5em"> | ||
| + | <FONT COLOR="red"> <b>Transformations and controls for future tests</b> </FONT> <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/> | 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/> | ||
| + | |||
| + | <b>Digestion</b> of pIG16 (E+P) and electrophoresis : the digestion had failed or the extracted plasmid was not pIG16.<br/> | ||
| + | <b>Digestion</b> 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/><br/> | ||
| + | </p> | ||
| + | |||
| + | <p style=" line-height : 1.5em"> | ||
| + | <FONT COLOR="purple"> <b>Adherence Test Preparation</b> </FONT> <br/><br/> | ||
Start of 5mL cultures for 24 well plates :<br/> | Start of 5mL cultures for 24 well plates :<br/> | ||
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LB : NM522/pIG16/p157, NM522/pIG16/p115, NM522/pIG16/p127, NM522/pIG16/p116, NM522<br/><br/> | LB : NM522/pIG16/p157, NM522/pIG16/p115, NM522/pIG16/p127, NM522/pIG16/p116, NM522<br/><br/> | ||
| - | + | ||
| - | + | ||
</p> | </p> | ||
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<h6 style="text-align :left"> Friday </h6><HR> | <h6 style="text-align :left"> Friday </h6><HR> | ||
| + | <br/> | ||
| + | |||
<p style=" line-height : 1.5em"> | <p style=" line-height : 1.5em"> | ||
| - | <b>Plating</b> of NM522/pIG16/p157, NM522/pIG16/p115, NM522/pIG16/p127, NM522/pIG16/p116 ( who have a double resistance ) on their second resistance ( Kan ).<br/> | + | <FONT COLOR="red"> <b>Transformations and controls for future tests</b> </FONT> <br/><br/> |
| + | <b>Plating</b> of NM522/pIG16/p157, NM522/pIG16/p115, NM522/pIG16/p127, NM522/pIG16/p116 (who have a double resistance) on their second resistance ( Kan ).<br/> | ||
<b>Extraction</b> (QIAgen) and <b>digestion</b>(Fermentas) of these plasmids by E : only one plasmid was in the strains -> the transformations need to be restarted.<br/><br/> | <b>Extraction</b> (QIAgen) and <b>digestion</b>(Fermentas) of these plasmids by E : only one plasmid was in the strains -> the transformations need to be restarted.<br/><br/> | ||
| + | |||
| + | |||
| + | 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.<br/><br/><br/> | ||
| + | |||
| + | </p> | ||
| + | |||
| + | <p style=" line-height : 1.5em"> | ||
| + | <FONT COLOR="purple"> <b>Adherence and Flocculation Test</b> </FONT> <br/><br/> | ||
Start of <b>24 well plates</b> :<br/> | Start of <b>24 well plates</b> :<br/> | ||
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<br> | <br> | ||
Start of tubes for flocculation : the same as plates (2) and (3).<br> | Start of tubes for flocculation : the same as plates (2) and (3).<br> | ||
| - | |||
| - | + | ||
</p> | </p> | ||
Revision as of 11:44, 15 September 2011
Week 9
From Monday the 15th of August to Friday the 19th of August 2011
Monday
Transformations and controls for future tests
Digestion with E+X, ligation with ...
Transformation and plating of NM522 with : pIG3 (3µL), pIG16 (3µL)
Tuesday
Transformations and controls for future tests
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.
Adherence Test
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
Wednesday
Transformations and controls for future tests
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.
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.
Adherence Test
Start of 24 well plate adherence test in M63G medium with PHL818 ( positive control ), NM522 ( negative control ), S18 + Amp + CoCl2 ( in increasing concentration )
Others
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 ).
Thursday
Transformations and controls for future tests
The transformation gave too many bacteria : plating of all the previous transformed strains on a new Petri dish in order to isolate individual clones.
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.
Adherence Test Preparation
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
Friday
Transformations and controls for future tests
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.
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.
Adherence and Flocculation Test
Start of 24 well plates :
(1) M63 : PHL818 ( positive control ), NM522 and NM522/pUC18( negative controls ), S18 + Co ( in increasing concentration)-> to characterize rcn-csgBAEFG
(2) M63 : PHL818, NM522, NM522/pUC18 (with and without cobalt), S17 and S18 ( with cobalt in increasing concentrations )-> to test the leak
(3) LB/2 : PHL818, NM522, NM522/pUC18 (with and without cobalt), S17 and S18 ( with cobalt in increasing concentrations )-> to test the leak
Start of tubes for flocculation : the same as plates (2) and (3).
