Difference between revisions of "Team:Paris Saclay/Notebook/July/5"

(→‎Lab work)
(→‎Tuesday 5th July)
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Thus this last gBlock  4.1 clone 3 was transformed again following the same protocol as the 21/06/2016 with :
 
Thus this last gBlock  4.1 clone 3 was transformed again following the same protocol as the 21/06/2016 with :
 
50μg of competent DH5α cells
 
50μg of competent DH5α cells
−
4.1 ligation product in 5μL of the plasmid puc19
+
4.1 ligation product in 5μL of the plasmid pUC19
 
4.1 clone 6 extracted DNA (which presented a good size strip on electrophoresis)
 
4.1 clone 6 extracted DNA (which presented a good size strip on electrophoresis)
  
 
6 clones of each of the other transformations [[Team:Paris_Saclay/Notebook/June/28#pPS16_001|pPS16_001, pPS16_002, pPS16_003, pPS16_005 and pPS16_006]] were grown in 4mL of LB with Ampicillin (50µg/mL) at 37°C, 180rpm.  For pPS16_002 only 2 clones were cultivated because there was not more colony. To obtain more colonies, another [[Team:Paris_Saclay/Experiments#HeatShockCompetent|transformation]] was made with 50µL of cells and 5µL of plasmid. 50µL of transformed bacteria were plated on LB + Ampicillin (50µg/mL).
 
6 clones of each of the other transformations [[Team:Paris_Saclay/Notebook/June/28#pPS16_001|pPS16_001, pPS16_002, pPS16_003, pPS16_005 and pPS16_006]] were grown in 4mL of LB with Ampicillin (50µg/mL) at 37°C, 180rpm.  For pPS16_002 only 2 clones were cultivated because there was not more colony. To obtain more colonies, another [[Team:Paris_Saclay/Experiments#HeatShockCompetent|transformation]] was made with 50µL of cells and 5µL of plasmid. 50µL of transformed bacteria were plated on LB + Ampicillin (50µg/mL).
  
 +
====Plasmid extraction====
 +
''By Laetitia and Alice'''
 +
The [[Team:Paris_Saclay/Experiments#PlasmidExtraction|usual protocol]] was used to extract plasmids pPS16_008 from 250μL of clone 3 overnight culture and pPS16_009 from 320μL of clone 1 overnight culture (from 28/06/16 transformation).
 +
Plasmids were resuspended in 10μL of water with RNAse (50μg/mL).
  
 
===Biobrick characterization===
 
===Biobrick characterization===
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|colspan="2"|1mM
 
|colspan="2"|1mM
 
|}
 
|}
 +
 +
Cultures were spun down for 5min at 1300rpm. Supernatant was discarded and glass marbles previously cleaned with 1M nitric acid were added with 50μL of Luc buffer. Tubes were vortexed for 30min at 4°C. Everything was then done on ice. 150μL of Luc bufer was added and cells were centrifuged for 15min at 13000rom at 4°C.
 +
 +
For the luciferase test, 4mL of Luc buffer were mixed to 80μL of ATP and 8μL of luciferine.
 +
For the βGal test, 400μL of Z buffer mixed with 10μL of extract and 100μL of ONPG were incubated for 8min at 30°C. Then 250μL of STOP buffer was added.
 +
OD<sub>420nm</sub> was measured.
  
 
[[File:T--Paris_Saclay--160705_characterization.jpeg|frame|Ratio between luciferase activity and β-Gal activity]]
 
[[File:T--Paris_Saclay--160705_characterization.jpeg|frame|Ratio between luciferase activity and β-Gal activity]]
  
 
{{Team:Paris_Saclay/notebook_footer}}
 
{{Team:Paris_Saclay/notebook_footer}}

Revision as of 09:26, 18 July 2016

Tuesday 5th July

Lab work

Visualization

Transformation of DH5α|pPS16_004 and DH5α|pPS16_007

By Caroline and Mathilde

The 04/06/2016 transformations show white colony growth for gBlocks 4.2 clone 3 and GFP1-9 (clone 1), but blue colonies only for 4.1 clone 3.

Thus this last gBlock 4.1 clone 3 was transformed again following the same protocol as the 21/06/2016 with : 50μg of competent DH5α cells 4.1 ligation product in 5μL of the plasmid pUC19 4.1 clone 6 extracted DNA (which presented a good size strip on electrophoresis)

6 clones of each of the other transformations pPS16_001, pPS16_002, pPS16_003, pPS16_005 and pPS16_006 were grown in 4mL of LB with Ampicillin (50µg/mL) at 37°C, 180rpm. For pPS16_002 only 2 clones were cultivated because there was not more colony. To obtain more colonies, another transformation was made with 50µL of cells and 5µL of plasmid. 50µL of transformed bacteria were plated on LB + Ampicillin (50µg/mL).

Plasmid extraction

By Laetitia and Alice' The usual protocol was used to extract plasmids pPS16_008 from 250μL of clone 3 overnight culture and pPS16_009 from 320μL of clone 1 overnight culture (from 28/06/16 transformation). Plasmids were resuspended in 10μL of water with RNAse (50μg/mL).

Biobrick characterization

B-Galactosidase and luciferase test on transformed BL21

By Charlene

Cultures tested

Plasmid(s) K1372001 K1372001 + pcl UAA K1372001 + pcl UAG K1372001 + pcl Tq
Clone 1 2 1 2 1 2 1 2 1 2 1 2 1 2 1 2 1 2 1 2 1 2 1 2
Salicylate concentration 0 30µM 1mM 0 30µM 1mM 0 30µM 1mM 0 30µM 1mM

Cultures were spun down for 5min at 1300rpm. Supernatant was discarded and glass marbles previously cleaned with 1M nitric acid were added with 50μL of Luc buffer. Tubes were vortexed for 30min at 4°C. Everything was then done on ice. 150μL of Luc bufer was added and cells were centrifuged for 15min at 13000rom at 4°C.

For the luciferase test, 4mL of Luc buffer were mixed to 80μL of ATP and 8μL of luciferine. For the βGal test, 400μL of Z buffer mixed with 10μL of extract and 100μL of ONPG were incubated for 8min at 30°C. Then 250μL of STOP buffer was added. OD420nm was measured.

Ratio between luciferase activity and β-Gal activity





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