Team:Evry/Notebook/CellCharacterization/Antibiotic test

From 2014.igem.org

(Difference between revisions)
Line 91: Line 91:
<tr >
<tr >
   <td> <b>Plates </b> </td>
   <td> <b>Plates </b> </td>
-
   <td> <b> LB Agar only </b> </td>
+
   <td>LB Agar only </td>
-
   <td> <b> LB+Cam </td> </b>
+
   <td>LB+Cam </td>
-
   <td> <b> LB+Kan </td> </b>
+
   <td>LB+Kan </td>
-
   <td> <b> LB+Amp </td> </b>
+
   <td>LB+Amp </td>
-
   <td> <b> LB+Erm </td> </b>
+
   <td>LB+Erm </td>
-
   <td> <b> LB+Tet </td> </b>
+
   <td>LB+Tet </td>
</tr>
</tr>
<tr>
<tr>
-
   <td>E.coli </td>
+
   <td> <b>E.coli</b> </td>
-
   <td> <b>  X  </td> </b>
+
   <td>X  </td>
-
   <td> <b>  0  </td> </b>
+
   <td>0  </td>
-
   <td> <b>  0  </td> </b>
+
   <td>0  </td>
-
   <td> <b>  X  </td> </b>
+
   <td>X  </td>
-
   <td> <b>  0  </td> </b>
+
   <td>0  </td>
</tr>
</tr>
</table>
</table>

Revision as of 09:47, 25 August 2014

Picture

Preparation of antibiotic stocks

Antibiotic Stock concentration Protocol
Kanamycin 25 mg/mL Weight 0.56g of Kanamycin powder, solubilization into 20mL of miliQ water.
Vortex 5min and filtration with a 200nm filter.
Stock : -20°C (aliquots of 1mL)

Streptomycin 100 mg/mL Weight 1g of Streptomycin powder, solubilization into 20mL of miliQ water.
Vortex 5min and filtration with a 200nm filter.
Stock : -20°C (aliquots of 1mL)

Amplicilin 50 mg/mL Weight 1g of Amplicilin powder, solubilization into 40mL of miliQ water.
Vortex 5min and filtration with a 200nm filter.
Stock : -20°C (aliquots of 1mL)

Tetracyclin 15 mg/mL Weight 0.6g of Tetracyclin powder, solubilization into 20mL of ethanol 50%.
Vortex 5min and filtration with a 200nm filter.
Stock : -20°C and hiden from light(aliquots of 1mL)
Chloramphenicol 34 mg/mL Weight 0.34g of Chloramphenicol powder, solubilization into 10mL of ethanol 100%.
Vortex 5min and filtration with a 200nm filter.
Stock : -20°C and hiden from light(aliquots of 1mL)

08-16-2014

Tests of antibiotics' stocks

Six plates of LB agar were made. Five of them contained one of those antibiotics in the dilution 1:1000:
  • Chloramphénicol
  • Kanamycin
  • Erythromycin
  • Ampicilin
  • Tetracyclin
(The last one was the control of the growth of our bacteria without antibiotics) We sowed 10µL of a liquid LB culture of Bl21, and we let them incubate overnight at 37°C.

Survivability tests

Two plates of MB 1X and M9 1X were made and divised in two parts. Each plate was sowed with 5µL of a liquid MB 1X culture of Pseudovibrio denitrificans(dated 12th August) in a part and with 5µL of a liquid M9 1X culture Pseudovibrio denitrificans (dated 12th August) in the other part.

Pre-cultures

Cultures in MB 1X and M9 1X of Pseudovibrio were passed by a 1/10 dilution and let incubate overnight at 30°C. New cultures of different E.Coli were also started from glycerol or plate and let incubated overnight at 37°C. From glycerol stocks:
  • Bl21
  • Top10
  • DH5a
From plates:
  • DH5a tranformed with pCB1C3
  • Top10 transformed with pQexp
  • DH5a pyr tranformed with pMK2
Bacteria tranformed with plasmid were cultivated with the corresponding antibiotic for the selection. For each medium we make a negative contrôle without bacteria.
08-17-2014

Tests of antibiotics' stocks - Results

Plates LB Agar only LB+Cam LB+Kan LB+Amp LB+Erm LB+Tet
E.coli X 0 0 X 0

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