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Team:Lyon-INSA-ENS/Realisation/Week12
From 2011.igem.org
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<p style=" line-height : 1.5em"> | <p style=" line-height : 1.5em"> | ||
| - | <b>TSS transformation</b> of MC4100 with pIG27 (Cm) or pIG16 (Amp). | + | <FONT COLOR="red"> <b>Transformations and controls for future tests</b> </FONT> <br/><br/> |
| + | <b>TSS transformation</b> of MC4100 with pIG27 (Cm) or pIG16 (Amp).<br/><br/> | ||
| - | <br><br> | + | <b>Digestion</b> and <b>electrophoresis</b> of the previously extracted DNA of MC4100+pIG6 clones and MC4100+pIG2 clones. Comparison made with the parts put in the collection.--> Verification is OK.<br/><br/><br/> |
| - | |||
| - | < | + | </p> |
| - | <b> | + | <p style=" line-height : 1.5em"> |
| + | <FONT COLOR="blue"> <b>Strain Collection</b> </FONT> <br/><br/> | ||
| + | Storage of the strains : <br/> | ||
| + | -<b>S25 = MC4100/piG6</b> (clone #3)<br/> | ||
| + | -<b>S26 = MC4100/piG2</b> (clone #3)<br/><br/><br/> | ||
| + | </p> | ||
| - | <br><br> | + | <p style=" line-height : 1.5em"> |
| + | <FONT COLOR="purple"> <b>Microscopy Test</b> </FONT> <br/><br/> | ||
| + | <b>Preparation</b><br/> | ||
| + | Start of 5mL liquid cultures of PHL1414/piG2 (AmpR), PHL1414/piG6 (AmpR) from solid cultures (09/02)<br/> | ||
| + | and S19/p127 (KanR/AmpR), S19/p116 (KanR/AmpR) from solid cultures (08/23).<br/><br/> | ||
| + | <b>Test</b><br/> | ||
| + | Preparation of plates for the following strains (two repetitions):<br/> | ||
| + | -S19/p127 KanR/AmpR<br/> | ||
| + | -S19/p116 KanR/AmpR = negative control<br/> | ||
| + | (18A-OmpR234 Characterization)<br/> | ||
| + | -PHL1414/piG6 AmpR = negative control without Co and with Co 10µM<br/> | ||
| + | -PHL1414/piG2 AmpR without Co and with Co 10µM, Co 25µM, Co 50µM, Co 100µM<br/> | ||
| + | (pRcn-csgBAEFG Characterization)<br/> | ||
| + | Incubation at 30°C overnight (for 15 hours).<br/><br/><br/> | ||
| + | |||
| + | </p> | ||
| + | |||
| + | <p style=" line-height : 1.5em"> | ||
| + | <FONT COLOR="purple"> <b>CFA Test</b> </FONT> <br/><br/> | ||
</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/> | ||
Results of tuesday transformations : no clone on the negative controls, several on the tests. Isolation of 4 clones per transformation in solid and liquid culture.<br/><br/> | Results of tuesday transformations : no clone on the negative controls, several on the tests. Isolation of 4 clones per transformation in solid and liquid culture.<br/><br/> | ||
| + | </p> | ||
| - | + | <p style=" line-height : 1.5em"> | |
| + | <FONT COLOR="purple"> <b>Results of Microscopy Test</b> </FONT> <br/><br/> | ||
| + | Observation of the different slides with a fluorescent microscope.<br/> | ||
| + | There is a problem with S19/p127 and S19/p116 so we start again the same test for these two strains.<br/><br/><br/> | ||
| + | </p> | ||
| + | |||
| + | <p style=" line-height : 1.5em"> | ||
| + | <FONT COLOR="purple"> <b>Results of Adherence Test - pRcn-csgBAEFG Characterization</b> </FONT> <br/><br/> | ||
| + | |||
| + | Revealing of the well plates started on Monday.<br/> | ||
| + | Measuring OD600 and Crystal Violet coloration.<br/><br/><br/> | ||
| + | </p> | ||
| + | |||
| + | <p style=" line-height : 1.5em"> | ||
| + | <FONT COLOR="orange"> <b>Others</b> </FONT> <br/><br/> | ||
| + | Pouring of Amp plates. | ||
| + | </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/> | ||
E+P <b>digestion and electrophoresis</b> of all the isolated clones for the following transformations : MC4100+pIG27, MC4100 + pIG16, MC4100 + rcn-ompR234.<br/> | E+P <b>digestion and electrophoresis</b> of all the isolated clones for the following transformations : MC4100+pIG27, MC4100 + pIG16, MC4100 + rcn-ompR234.<br/> | ||
All clones are correct for pIG27 and pIG16. No clone is correct for rcn-ompR234 -> detection of an error in the ligation. <br/> | All clones are correct for pIG27 and pIG16. No clone is correct for rcn-ompR234 -> detection of an error in the ligation. <br/> | ||
Revision as of 13:30, 15 September 2011
Week 12
From Monday the 5th of September to Friday the 9th of September 2011
Monday
Transformations and controls for future tests
Miniprep of three individual clones of MC4100/piG6 and MC4100/piG2 isolated on 09/02 in order to chek the presence of the right plasmid.
Results of Flocculation Test and New Test
Crystal Violet coloration for the series of 09/02.
Start of a fourth test in M63G medium with :
-MC4100/piG6 (AmpR) without Co and with Co 10µM (negative controls)
-MC4100/piG2 (AmpR) without Co and with Co 10µM, 25µM, 50µM, 100µM.
(three repetitions)
These liquid cultures are let for two days at 30°C and low stirring (75).
Adherence Test – pRcn-csgBAEFG Characterization
Start of three replica of 24 well plates in M63G medium + Amp with MC4100/piG6 without Co and with Co 10µM
(negative control) and MC4100/piG2 without Co and with Co 10µM, 25µM, 50µM, 100µM.
Incubation at 30°C for 48h.
Revealing of the well plates started on 09/02.
Measuring OD600 and Crystal Violet coloration.
Tuesday
Transformations and controls for future tests
TSS transformation of MC4100 with pIG27 (Cm) or pIG16 (Amp).
Digestion and electrophoresis of the previously extracted DNA of MC4100+pIG6 clones and MC4100+pIG2 clones. Comparison made with the parts put in the collection.--> Verification is OK.
Strain Collection
Storage of the strains :
-S25 = MC4100/piG6 (clone #3)
-S26 = MC4100/piG2 (clone #3)
Microscopy Test
Preparation
Start of 5mL liquid cultures of PHL1414/piG2 (AmpR), PHL1414/piG6 (AmpR) from solid cultures (09/02)
and S19/p127 (KanR/AmpR), S19/p116 (KanR/AmpR) from solid cultures (08/23).
Test
Preparation of plates for the following strains (two repetitions):
-S19/p127 KanR/AmpR
-S19/p116 KanR/AmpR = negative control
(18A-OmpR234 Characterization)
-PHL1414/piG6 AmpR = negative control without Co and with Co 10µM
-PHL1414/piG2 AmpR without Co and with Co 10µM, Co 25µM, Co 50µM, Co 100µM
(pRcn-csgBAEFG Characterization)
Incubation at 30°C overnight (for 15 hours).
CFA Test
Wednesday
Transformations and controls for future tests
Results of tuesday transformations : no clone on the negative controls, several on the tests. Isolation of 4 clones per transformation in solid and liquid culture.
Results of Microscopy Test
Observation of the different slides with a fluorescent microscope.
There is a problem with S19/p127 and S19/p116 so we start again the same test for these two strains.
Results of Adherence Test - pRcn-csgBAEFG Characterization
Revealing of the well plates started on Monday.
Measuring OD600 and Crystal Violet coloration.
Others
Pouring of Amp plates.
Thursday
Transformations and controls for future tests
E+P digestion and electrophoresis of all the isolated clones for the following transformations : MC4100+pIG27, MC4100 + pIG16, MC4100 + rcn-ompR234.
All clones are correct for pIG27 and pIG16. No clone is correct for rcn-ompR234 -> detection of an error in the ligation.
Storage of clone 1 for pIG27 and pIG16. Plating of those same clones on LB+antibiotic (Cm and Amp respectively ).
Friday
Nanodrop quantification of the previous pIG27 miniprep : 48.0 ng/µL.
Preparation of tubes for sequencing (30µL at approximately 50-100 ng/µL). The pIG27 miniprep is used as such. pIG25 is diluted from 5µL of the collection (371.4 ng/µL) with 25µL water.
