Difference between revisions of "Team:Paris Saclay/Notebook/July/4"

(→‎Digestion of plasmids)
(→‎Lab work)
 
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{{Team:Paris_Saclay/notebook_header}}
 
{{Team:Paris_Saclay/notebook_header}}
 
=Monday 4<sup>th</sup> July=
 
=Monday 4<sup>th</sup> July=
−
==Lab work==
 
  
 
===Visualization===
 
===Visualization===
 +
====[[Team:Paris_Saclay/Experiments#Ligation|Ligation]] of gBlock 2.2====
 +
''By Caroline and Lea''
 +
 +
The size of the gBlock 2.2 is 808bp and the ratio was 11.69 (>7).
 +
gBlock was gently centrifugated and resuspended with 75µL of TE. The solution was then vortexed and stored at 50°C for 20 min. 1µL of ligase buffer, 1µL of ligase and 1µL of digested plasmids pUC19 were mixed with 7µL of gBlock 2.2. The resulting plasmid will be called pPS16_004.
 +
<div id="pPS16_004"></div>
 +
 +
2 control solutions were made:
 +
# 1µL of digested plasmids pUC19 and 9µL of water
 +
# 1µL of plasmid, 1µL of ligase and 1µL of water.
 +
 +
====gBlocks transformation in DH5α====
 +
''By Caroline and Lea''
 +
 +
6 transformations were performed with the ligation products containing plasmids: [[Team:Paris_Saclay/Notebook/June/28#pPS16_001|pPS16_001, pPS16_002, pPS16_003, pPS16_005, pPS16_006]] and [[#pPS16_004|pPS16_004]] carried out on the morning. 3 other transformations were performed with plasmids [[Team:Paris_Saclay/Notebook/June/28#pPS16_001|pPS16_007, pPS16_008 and pPS16_009]] extracted on 30/06/16. The [[Team:Paris_Saclay/Experiments#HeatShockCompetent|transformation protocol]] was followed increasing plasmids quantities (5µL instead of 1µL).
 +
Afterward cells were spread on Petri dishes containing LB + Ampicillin (50µg/mL) + X-Gal + IPTG.
 +
 
====Digestion of plasmids====
 
====Digestion of plasmids====
 
''By Mathilde and Alice''
 
''By Mathilde and Alice''
  
−
The following plasmids were digested again with EcoR1 and HindIII using [[#PlasmidDigestion|this protocol]]:
+
The following plasmids were [[Team:Paris_Saclay/Experiments#PlasmidDigestion|digested]] again with EcoRI and HindIII:
 
*pPS16_001 (from the 6 clones that were selected on 29/06/16)
 
*pPS16_001 (from the 6 clones that were selected on 29/06/16)
 
*pPS16_003 (from the 6 clones that were selected on 29/06/16)
 
*pPS16_003 (from the 6 clones that were selected on 29/06/16)
Line 34: Line 50:
 
|}
 
|}
  
−
The mix was incubated at 37°C for 1 hour.
+
The digestion products were mixed as follow to be migrated on an agarose gel.
−
An electrophoresis was done (0.8% of agarose).
+
[[File:T--Paris_Saclay--160704_visualizatio_gBlocks_échelle1.jpg|400px|thumb|right|pPS16_001, pPS16_003, pPS16_005 and pPS16_006 Migration]]
−
 
+
 
{| class="wikitable"
 
{| class="wikitable"
 
|-
 
|-
Line 49: Line 64:
 
|}
 
|}
  
−
The DNA concentration was still insufficient meaning the extraction step was not efficient.
+
The DNA concentration was still insufficient meaning the extraction steps were not efficient.
  
 
===Bringing DNA closer===
 
===Bringing DNA closer===
Line 55: Line 70:
 
''By Naiane and Laetitia''
 
''By Naiane and Laetitia''
  
−
Plasmids from the following strains were extracted with the kit "Charge Switch-Pro Plasmid Miniprep":
+
The following plasmids were extracted using the [[Team:Paris_Saclay/Experiments#InvitrogenPlasmidExtraction|"Charge Switch-Pro Plasmid Miniprep"]] kit:
−
*DS-NMCas
+
*DS-NMcas
−
*DS-SPCasN
+
*DS-SPcasN-
−
*DS-ST1
+
*DS-ST1casN-
−
*DS-TDCasN
+
*DS-TDcasN-
−
Then plasmids were digested with AvrII  
+
Then plasmids were [[Team:Paris_Saclay/Experiments#PlasmidDigestion|digested]] with AvrII :
  
 
{| class="wikitable"
 
{| class="wikitable"
Line 98: Line 113:
 
|}
 
|}
  
 +
===BioBrick K1372001 characterization===
 +
 +
====Preparation of salycilate at 10 mM====
 +
''By Charlène, Naiane and Léa''
 +
 +
0.07g of salycilic acid was diluted in 50mL of water. The pH was adjusted to 7 with NaOH.
 +
 +
====Culture of BL21====
 +
''By Lea and Caroline''
 +
 +
2 colonies from each Petri dish were grown in 500µL of LB, streptomycin (50µg/mL) and chloramphenicol (30µg/mL).
 +
Then each solution was split into 3x150µL and 350µL of LB + streptomycin (50µg/mL) + chloramphenicol (30µg/mL) were added to each culture.
 +
Salicylate (main solution at 10mM) was added to obtain the following concentrations :
 +
 +
{| class="wikitable"
 +
|-
 +
!Plasmid(s)
 +
|colspan="6"|K1372001
 +
 +
|colspan="6"|K1372001 + pcl_TAA
 +
|colspan="6"|K1372001 + pcl_TAG
 +
|colspan="6"|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
 +
|colspan="2"|0
 +
|colspan="2"|30µM
 +
|colspan="2"|1mM
 +
|colspan="2"|0
 +
|colspan="2"|30µM
 +
|colspan="2"|1mM
 +
|colspan="2"|0
 +
|colspan="2"|30µM
 +
|colspan="2"|1mM
 +
|colspan="2"|0
 +
|colspan="2"|30µM
 +
|colspan="2"|1mM
 +
|}
  
 
{{Team:Paris_Saclay/notebook_footer}}
 
{{Team:Paris_Saclay/notebook_footer}}

Latest revision as of 14:05, 9 October 2016

Monday 4th July

Visualization

Ligation of gBlock 2.2

By Caroline and Lea

The size of the gBlock 2.2 is 808bp and the ratio was 11.69 (>7). gBlock was gently centrifugated and resuspended with 75µL of TE. The solution was then vortexed and stored at 50°C for 20 min. 1µL of ligase buffer, 1µL of ligase and 1µL of digested plasmids pUC19 were mixed with 7µL of gBlock 2.2. The resulting plasmid will be called pPS16_004.

2 control solutions were made:

  1. 1µL of digested plasmids pUC19 and 9µL of water
  2. 1µL of plasmid, 1µL of ligase and 1µL of water.

gBlocks transformation in DH5α

By Caroline and Lea

6 transformations were performed with the ligation products containing plasmids: pPS16_001, pPS16_002, pPS16_003, pPS16_005, pPS16_006 and pPS16_004 carried out on the morning. 3 other transformations were performed with plasmids pPS16_007, pPS16_008 and pPS16_009 extracted on 30/06/16. The transformation protocol was followed increasing plasmids quantities (5µL instead of 1µL). Afterward cells were spread on Petri dishes containing LB + Ampicillin (50µg/mL) + X-Gal + IPTG.

Digestion of plasmids

By Mathilde and Alice

The following plasmids were digested again with EcoRI and HindIII:

  • pPS16_001 (from the 6 clones that were selected on 29/06/16)
  • pPS16_003 (from the 6 clones that were selected on 29/06/16)
  • pPS16_005 (from the 6 clones that were selected on 29/06/16)
  • pPS16_006 (from the 6 clones that were selected on 29/06/16)
Component Volume (µL)
Plasmid 10
Red buffer 10x 2
Water 7
EcoRI enzyme 0.5
HindIII enzyme 0.5

The digestion products were mixed as follow to be migrated on an agarose gel.

pPS16_001, pPS16_003, pPS16_005 and pPS16_006 Migration
Component Volume (µL)
Digested DNA 20
Loading buffer 3.3

The DNA concentration was still insufficient meaning the extraction steps were not efficient.

Bringing DNA closer

Plasmids extraction

By Naiane and Laetitia

The following plasmids were extracted using the "Charge Switch-Pro Plasmid Miniprep" kit:

  • DS-NMcas
  • DS-SPcasN-
  • DS-ST1casN-
  • DS-TDcasN-

Then plasmids were digested with AvrII :

Component Volume (µL)
Plasmid 10
Tango buffer 10x 2
Water 7
AvrII enzyme 1

The mix was incubated at 37°C for 1 hour. An electrophoresis was done (0.8% of agarose).

Component Volume (µL)
Digested DNA 5
Water 5
Loading buffer 2

BioBrick K1372001 characterization

Preparation of salycilate at 10 mM

By Charlène, Naiane and Léa

0.07g of salycilic acid was diluted in 50mL of water. The pH was adjusted to 7 with NaOH.

Culture of BL21

By Lea and Caroline

2 colonies from each Petri dish were grown in 500µL of LB, streptomycin (50µg/mL) and chloramphenicol (30µg/mL). Then each solution was split into 3x150µL and 350µL of LB + streptomycin (50µg/mL) + chloramphenicol (30µg/mL) were added to each culture. Salicylate (main solution at 10mM) was added to obtain the following concentrations :

Plasmid(s) K1372001 K1372001 + pcl_TAA K1372001 + pcl_TAG 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





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