Difference between revisions of "Team:Paris Saclay/Notebook/June/30"

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{{Team:Paris_Saclay/notebook_header}}
 
{{Team:Paris_Saclay/notebook_header}}
 
=Thursday 30<sup>th</sup> June=
 
=Thursday 30<sup>th</sup> June=
−
==Lab work==
 
  
 
===Visualization===
 
===Visualization===
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''By Alice and Lea''
 
''By Alice and Lea''
  
−
DNA from bacteria transformed with plasmids containing gBlocks was extracted following the extraction protocol. For each gblock, DNA of 6 different clones was extracted.
+
Plasmids [[Team:Paris_Saclay/Notebook/June/28#pPS16_001|pPS16_001-009]] (except pPS16_004) were extracted following the [[Team:Paris_Saclay/Experiments#PlasmidExtraction|extraction protocol]]. Each plasmids were extracted from 6 different clones of bacteria.
−
At the end, plamids were resuspended in water/RNAse (5µL RNAse 10 mg/mL for 1 mL of water) to send to sequencing.
+
At the end, plasmids were resuspended in water/RNAse (5µL RNAse 10 mg/mL for 1 mL of water).
 +
 
 +
====Digestion of the plasmids containing gBlocks====
 +
''By Alice and Léa''
 +
 
 +
After extraction, plasmids were [[Team:Paris_Saclay/Experiments#PlasmidDigestion|digested]] with EcoRI and HindIII.
 +
 
 +
{| class="wikitable"
 +
! Component
 +
! Volume (µL)
 +
|-
 +
| Plasmids
 +
| 3
 +
|-
 +
| Red buffer 10x
 +
| 2
 +
|-
 +
| Water
 +
| 14
 +
|-
 +
| EcoRI enzyme
 +
| 0.5
 +
|-
 +
| HindIII enzyme
 +
| 0.5
 +
|}
 +
 
 +
The mix was incubated for 1 hour at 37°C and then stored at -20°C until the gel electrophoresis was done. Fragments are expected to be as follow:
 +
 
 +
{| class="wikitable"
 +
|-
 +
!Plasmid name
 +
!Plasmid size (kb)
 +
!Digestion product size (kb)
 +
|-
 +
|[[Team:Paris_Saclay/Notebook/June/28#pPS16_001|pPS16_001]]
 +
|3.7
 +
|2.7 and 1
 +
|-
 +
|[[Team:Paris_Saclay/Notebook/June/28#pPS16_002|pPS16_002]]
 +
|3.7
 +
|2.7 and 1
 +
|-
 +
|[[Team:Paris_Saclay/Notebook/June/28#pPS16_003|pPS16_003]]
 +
|3.7
 +
|2.7 and 1
 +
|-
 +
|[[Team:Paris_Saclay/Notebook/June/28#pPS16_005|pPS16_005]]
 +
|3.7
 +
|2.7 and 1
 +
|-
 +
|[[Team:Paris_Saclay/Notebook/June/28#pPS16_006|pPS16_006]]
 +
|3.7
 +
|2.7 and 1
 +
|-
 +
|[[Team:Paris_Saclay/Notebook/June/28#pPS16_007|pPS16_007]]
 +
|3.4
 +
|2.7 and 0.7
 +
|-
 +
|[[Team:Paris_Saclay/Notebook/June/28#pPS16_008|pPS16_008]]
 +
|4
 +
|2.7 and 1.3
 +
|-
 +
|[[Team:Paris_Saclay/Notebook/June/28#pPS16_009|pPS16_009]]
 +
|3.6
 +
|2.7 and 0.9
 +
|}
 +
 
 +
===Biobrick characterization===
 +
====BL21 electrocompetent cells preparation and transformation====
 +
''By Caroline and Charlene''
 +
 
 +
The [[Team:Paris_Saclay/Experiments#ElectroCompetent|usual protocol]] was used for transforming cells with K1372001, K1372001+pcl_TAA and the controls which were done following the same protocol but without plasmids.
 +
K1372001 and the controls were streaked on LB + Cm (30µg/mL). K1372001+pcl_TAA was streaked on LB + Cm (30µg/mL) + streptomicin (50µg/mL).
 +
This time two different streaks were made for each plasmid, the first one using 50µL from the culture, the second one with 400µL concentrated by centrifugation.
 +
 
 +
The Petri dishes made on 29/06 were stored at 4ºC. 
 +
 
  
 
{{Team:Paris_Saclay/notebook_footer}}
 
{{Team:Paris_Saclay/notebook_footer}}

Latest revision as of 13:52, 9 October 2016

Thursday 30th June

Visualization

Extraction of the plasmids containing gBlocks

By Alice and Lea

Plasmids pPS16_001-009 (except pPS16_004) were extracted following the extraction protocol. Each plasmids were extracted from 6 different clones of bacteria. At the end, plasmids were resuspended in water/RNAse (5µL RNAse 10 mg/mL for 1 mL of water).

Digestion of the plasmids containing gBlocks

By Alice and Léa

After extraction, plasmids were digested with EcoRI and HindIII.

Component Volume (µL)
Plasmids 3
Red buffer 10x 2
Water 14
EcoRI enzyme 0.5
HindIII enzyme 0.5

The mix was incubated for 1 hour at 37°C and then stored at -20°C until the gel electrophoresis was done. Fragments are expected to be as follow:

Plasmid name Plasmid size (kb) Digestion product size (kb)
pPS16_001 3.7 2.7 and 1
pPS16_002 3.7 2.7 and 1
pPS16_003 3.7 2.7 and 1
pPS16_005 3.7 2.7 and 1
pPS16_006 3.7 2.7 and 1
pPS16_007 3.4 2.7 and 0.7
pPS16_008 4 2.7 and 1.3
pPS16_009 3.6 2.7 and 0.9

Biobrick characterization

BL21 electrocompetent cells preparation and transformation

By Caroline and Charlene

The usual protocol was used for transforming cells with K1372001, K1372001+pcl_TAA and the controls which were done following the same protocol but without plasmids. K1372001 and the controls were streaked on LB + Cm (30µg/mL). K1372001+pcl_TAA was streaked on LB + Cm (30µg/mL) + streptomicin (50µg/mL). This time two different streaks were made for each plasmid, the first one using 50µL from the culture, the second one with 400µL concentrated by centrifugation.

The Petri dishes made on 29/06 were stored at 4ºC.






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