Team:Lyon-INSA-ENS/Realisation/Week13

From 2011.igem.org

(Difference between revisions)
Line 51: Line 51:
MC4100 + pIG34 (S32) <br/><br/>
MC4100 + pIG34 (S32) <br/><br/>
-
Storage of rcn-OmpR in psB1K3 (pIG34) from the previous miniprep.
+
Storage of rcn-OmpR in psB1K3 (pIG34) from the previous miniprep. <br/><br/>
</p>
</p>
Line 66: Line 66:
Dilution of 5µL pIG25 (371.4 ng/µL) into 25µL water to obtain 3 additional tubes at concentration of about 50ng/µL for sequencing. <br/>
Dilution of 5µL pIG25 (371.4 ng/µL) into 25µL water to obtain 3 additional tubes at concentration of about 50ng/µL for sequencing. <br/>
-
Sequencing (pIG25 and pIG27) sent.
+
Sequencing (pIG25 and pIG27) sent. <br/><br/>
 +
 
 +
</p>
 +
 
 +
<p style=" line-height : 1.5em">
 +
<FONT COLOR="purple"> <b> Microscopy Test </b> </FONT> <br/><br/>
 +
 
 +
<b>Preparation</b><br/>
 +
Sterilization of microscope cover slips in ethanol during a few minutes, then rinsed in sterilized water. <br/><br/>
 +
 
 +
<b>Test</b> <br/><br/>
 +
 
 +
Preparation of plates for the following strains (one repetition):<br/>
 +
-PHL1414/piG3 AmpR = negative control without Co and with Co 25µM<br/>
 +
-PHL1414/piG6 AmpR without Co, Co 10µM, Co 25µM, Co 50µM and Co 100µM<br/><br/>
 +
 
 +
Incubation at 30°C overnight (during 16 hours).<br/><br/><br/>
 +
 
</p>
</p>

Revision as of 15:15, 15 September 2011









Week 13


From Monday the 12th of September to Friday the 16th of September 2011







Monday


Transformations and controls for future tests

Plasmid and strain Collection

Plating of S17 from the collection to extract a bigger quantity of pIG25 ( pSB1C3 containing rcn-csgBAEFG )

Storage of the correct clone of:
PHL1414 + pIG3 (S30)
PHL1414+pIG16 (S31)
MC4100 + pIG34 (S32)

Storage of rcn-OmpR in psB1K3 (pIG34) from the previous miniprep.

Adherence Test Preparation

Others

Pouring of 9 LB+Amp plates

Dilution of 5µL pIG25 (371.4 ng/µL) into 25µL water to obtain 3 additional tubes at concentration of about 50ng/µL for sequencing.
Sequencing (pIG25 and pIG27) sent.

Microscopy Test

Preparation
Sterilization of microscope cover slips in ethanol during a few minutes, then rinsed in sterilized water.

Test

Preparation of plates for the following strains (one repetition):
-PHL1414/piG3 AmpR = negative control without Co and with Co 25µM
-PHL1414/piG6 AmpR without Co, Co 10µM, Co 25µM, Co 50µM and Co 100µM

Incubation at 30°C overnight (during 16 hours).




Tuesday



Transformations and controls for future tests

Plasmid and strain Collection

Start of a 5mL liquid culture of S17 in LB+Cm medium
Minipreps of this culture to extract pIG25 in larger quantity. Digestion (E+P) and electrophoresis for control : the insert is too small, not correct.

Adherence Test Preparation

Others





Wednesday


Transformations and controls

The Ptrc strong promoter has been received from Genecust in pBluescriptIISK+: transformation in MC4100 and plating on LB+Amp medium.

Digestion by E+P of this plasmid and ligation into pSB1C3 overnight

Plating of PHL916 on LB ( sureE strain that should not recombine DNA ) in case the transformation of pIG25 into MC4100 fails.

Plasmid and strain Collection

rcn-OmpR234 adherence test

Start of a rcn-OmpR234 24 well plate with :
S27 ( rcn in pSB1C3 ) without cobalt and with cobalt (25µM)
S33 ( rcn-OmpR234 in pSB1C3 ) with increasing concentration of cobalt (0, 10, 25 and 100 µM)

Others

Plating of PHL644, PHL1256, PHL628 to be sent to other research teams, on request.





Thursday




Friday








ENS assystem Biomérieux INSA INSA