Difference between revisions of "Team:Paris Saclay/Notebook/June/29"

(→‎Plasmids digestion)
(→‎Lab work)
 
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{{Team:Paris_Saclay/notebook_header}}
 
{{Team:Paris_Saclay/notebook_header}}
 
=Wednesday 29<sup>th</sup> June=
 
=Wednesday 29<sup>th</sup> June=
−
==Lab work==
 
  
 
===Visualization===
 
===Visualization===
−
====Culture of clones with gBlocks ====  
+
====Culture of clones containing the gBlocks ====  
 
''By Lea and Marion''
 
''By Lea and Marion''
  
−
6 clones of each construction were selected and grown overnight in 3.5mL of LB (50µg/ml Amp) at 37°C, 180rpm.
+
6 clones of each construction were selected (transformed cells with the plasmid containing the gBlocks are supposed to grow white on xGal/IPTG) and grown overnight in 3.5mL of LB (50µg/ml Amp) at 37°C, 180rpm.
  
  
 
===BioBrick characterization===
 
===BioBrick characterization===
−
====BL21 electro-competent cells preparation====
+
====BL21 electro-competent cells [[Team:Paris_Saclay/Experiments#ElectroCompetent|preparation and transformation]]====
 
''By Charlene''
 
''By Charlene''
  
−
200µL of BL21 pre-culture was added to 15mL of LB and incubated for 4h at 37°C. When abs<sub>600nm</sub>=0.68, cells were centrifuged for 10 minutes at 4000rpm and washed twice with addition of 10mL glycerol (10%) followed by 10 minutes of centrifugation at 4000rpm. Cells were mixed with 200µL glycerol and kept -80°C.  
+
200µL of BL21 pre-culture was added to 15mL of LB and incubated for 4h at 37°C. When OD<sub>600nm</sub>=0.68, cells were centrifuged for 10 minutes at 4000rpm and washed twice with addition of 10mL glycerol (10%) followed by 10 minutes of centrifugation at 4000rpm. Cells were mixed with 200µL glycerol and kept -80°C.
  
−
====BL21 electro-competent cells transformation====
+
{| class="wikitable"
−
''By Charlene''
+
|-
 +
!Plasmids used
 +
!Description
 +
!Use
 +
|-
 +
|pcl_TAA
 +
|Promoteur_RBS_LacZ_TAA_Luciferase_Terminator
 +
|Negative control = base level
 +
|-
 +
|pcl_TAG
 +
|Promoteur_RBS_LacZ_TAG_Luciferase_Terminator
 +
|Our interest plasmids allowing us to measured the biobrick activity
 +
|-
 +
|pcl_Tq
 +
|Promoteur_RBS_LacZ_CodonSens_Luciferase_Terminator
 +
|Positive control = luciferase 100% activity
 +
|}
  
 
We realized four transformations with the following plasmids:
 
We realized four transformations with the following plasmids:
Line 26: Line 41:
 
50µL of cells were added in the cuvettes with 1µL DNA (diluted 1/10). Everything was done at 4°C.  
 
50µL of cells were added in the cuvettes with 1µL DNA (diluted 1/10). Everything was done at 4°C.  
  
−
Transformation did not work for K1372001 and K1372001+pcl_TAA because the cuvettes were broken. {{Font color||yellow|Value = 1.6 What is this value???}}. Cells died during transformation.
+
Transformation did not work for K1372001 and K1372001+pcl_TAA because the cuvettes were broken (electroporation time constant was equal to 1.6ms. Cells died during transformation.
−
Transformation worked for K1372001+pcl_TAG and K1372001+pcl_Tq. {{Font color||yellow|Value = 6}}.  We added 1mL of LB to the cells and incubated them for 1h at 37°C.
+
Transformation worked for K1372001+pcl_TAG and K1372001+pcl_Tq (time constant was equal to 6ms.  We added 1mL of LB to the cells and incubated them for 1h at 37°C.
  
 
The following cells were plated on Petri dishes (30µg/mL Cm + 50µg/mL streptomycin):
 
The following cells were plated on Petri dishes (30µg/mL Cm + 50µg/mL streptomycin):
Line 33: Line 48:
 
*BL21|K1372001+pcl_TAG : 500µL of transformed cells concentrated 5x.
 
*BL21|K1372001+pcl_TAG : 500µL of transformed cells concentrated 5x.
 
*BL21|K1372001+pcl_Tq : 50µL cells
 
*BL21|K1372001+pcl_Tq : 50µL cells
−
*BL21|K1372001+pcl Tq : 500µL of transformed cells concentrated 5x.
+
*BL21|K1372001+pcl_Tq : 500µL of transformed cells concentrated 5x.
−
 
+
  
 
====K1372001 plasmid digestion====
 
====K1372001 plasmid digestion====
 
''By Naiane''
 
''By Naiane''
  
−
K1372001 plasmid was digested with BglI using the following quantities: 3 µL DNA, 2µL buffer (O), 1 µL enzyme (BglI), 14µL water. The mix was incubated for 1h at 37°C and 10 min at 55°C and kept at -20°C until the gel electrophoresis was done. Fragments are expected to size 1.2kb and 2.4kb.
+
K1372001 plasmid was [[Team:Paris_Saclay/Experiments#PlasmidDigestion|digested with BglI]] using the following quantities: 3 µL DNA, 2µL buffer (O), 1 µL enzyme (BglI), 14µL water. The mix was incubated for 1h at 37°C and 10 min at 55°C and kept at -20°C until the gel electrophoresis was done. Fragments are expected to size 1.2kb and 2.4kb.
  
 +
====BL21 cell culture====
 +
We added 200µL of the BL21 culture made on 28/06 in 15ml of LB and incubated it overnight at 37°C, 180rpm. We need a new BL21 culture to redo the experiment because two of the transformations (K1372001 and pcl_TAA) did not work out (broken electroporation cuvettes).
  
 
===Bringing DNA closer===
 
===Bringing DNA closer===
 
====Plasmids digestion====
 
====Plasmids digestion====
 
''By Naiane''
 
''By Naiane''
−
Plasmids received from Addgene were digested with AvrII using the following quantities: 5 µL DNA, 2µL buffer (Tango), 1 µL enzyme (AvrII), 12µL water. The mix was incubated for 1h at 37°C and 10 min at 55°C and kept at -20°C until the gel electrophoresis was done. Fragments are expected to be as follow:
+
 
 +
Plasmids received from Addgene were [[Team:Paris_Saclay/Experiments#PlasmidDigestion|digested with AvrII]].
 +
 
 +
{| class="wikitable"
 +
|-
 +
! Component
 +
! Volume (µL)
 +
|-
 +
| Plasmid
 +
| 5
 +
|-
 +
| Tango buffer
 +
| 2
 +
|-
 +
| Water
 +
| 12
 +
|-
 +
| AvrII enzyme
 +
| 1
 +
|}
 +
 
 +
The mix was incubated for 1h at 37°C and 10 min at 55°C and kept at -20°C until the gel electrophoresis was done. Fragments are expected to be as follow:
 
{| class="wikitable"
 
{| class="wikitable"
 
|-
 
|-
 
!Plasmid name
 
!Plasmid name
 
!Plasmid size (kb)
 
!Plasmid size (kb)
−
!Digestion product size (kb)
+
!Expected digestion product size (kb)
 
|-
 
|-
 
|DS-NMcas
 
|DS-NMcas
Line 67: Line 104:
 
|6.9
 
|6.9
 
|2.2 and 4.6
 
|2.2 and 4.6
−
|-
+
|}
 +
 
 +
The migration's result was:
  
 +
[[File:T--Paris_Saclay--160629_bringingDNAcloser_casdigestion_échelle.JPG|200px|thumb|left|TD,NM,SP,ST1 electrophoresis]]
  
 
{{Team:Paris_Saclay/notebook_footer}}
 
{{Team:Paris_Saclay/notebook_footer}}

Latest revision as of 13:24, 9 October 2016

Wednesday 29th June

Visualization

Culture of clones containing the gBlocks

By Lea and Marion

6 clones of each construction were selected (transformed cells with the plasmid containing the gBlocks are supposed to grow white on xGal/IPTG) and grown overnight in 3.5mL of LB (50µg/ml Amp) at 37°C, 180rpm.


BioBrick characterization

BL21 electro-competent cells preparation and transformation

By Charlene

200µL of BL21 pre-culture was added to 15mL of LB and incubated for 4h at 37°C. When OD600nm=0.68, cells were centrifuged for 10 minutes at 4000rpm and washed twice with addition of 10mL glycerol (10%) followed by 10 minutes of centrifugation at 4000rpm. Cells were mixed with 200µL glycerol and kept -80°C.

Plasmids used Description Use
pcl_TAA Promoteur_RBS_LacZ_TAA_Luciferase_Terminator Negative control = base level
pcl_TAG Promoteur_RBS_LacZ_TAG_Luciferase_Terminator Our interest plasmids allowing us to measured the biobrick activity
pcl_Tq Promoteur_RBS_LacZ_CodonSens_Luciferase_Terminator Positive control = luciferase 100% activity

We realized four transformations with the following plasmids:

  • K1372001
  • K1372001+pcl_TAA
  • K1372001+pcl_TAG
  • K1372001+pcl_Tq

50µL of cells were added in the cuvettes with 1µL DNA (diluted 1/10). Everything was done at 4°C.

Transformation did not work for K1372001 and K1372001+pcl_TAA because the cuvettes were broken (electroporation time constant was equal to 1.6ms. Cells died during transformation. Transformation worked for K1372001+pcl_TAG and K1372001+pcl_Tq (time constant was equal to 6ms. We added 1mL of LB to the cells and incubated them for 1h at 37°C.

The following cells were plated on Petri dishes (30µg/mL Cm + 50µg/mL streptomycin):

  • BL21|K1372001+pcl_TAG : 50µL of transformed cells
  • BL21|K1372001+pcl_TAG : 500µL of transformed cells concentrated 5x.
  • BL21|K1372001+pcl_Tq : 50µL cells
  • BL21|K1372001+pcl_Tq : 500µL of transformed cells concentrated 5x.

K1372001 plasmid digestion

By Naiane

K1372001 plasmid was digested with BglI using the following quantities: 3 µL DNA, 2µL buffer (O), 1 µL enzyme (BglI), 14µL water. The mix was incubated for 1h at 37°C and 10 min at 55°C and kept at -20°C until the gel electrophoresis was done. Fragments are expected to size 1.2kb and 2.4kb.

BL21 cell culture

We added 200µL of the BL21 culture made on 28/06 in 15ml of LB and incubated it overnight at 37°C, 180rpm. We need a new BL21 culture to redo the experiment because two of the transformations (K1372001 and pcl_TAA) did not work out (broken electroporation cuvettes).

Bringing DNA closer

Plasmids digestion

By Naiane

Plasmids received from Addgene were digested with AvrII.

Component Volume (µL)
Plasmid 5
Tango buffer 2
Water 12
AvrII enzyme 1

The mix was incubated for 1h at 37°C and 10 min at 55°C and kept at -20°C until the gel electrophoresis was done. Fragments are expected to be as follow:

Plasmid name Plasmid size (kb) Expected digestion product size (kb)
DS-NMcas 6 2.2 and 3.7
DS-SPcasN- 6.8 2.2 and 4.5
DS-ST1casN- 6 2.2 and 3.8
DS-TDcasN- 6.9 2.2 and 4.6

The migration's result was:

TD,NM,SP,ST1 electrophoresis





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