Difference between revisions of "Team:Paris Saclay/Notebook/July/21"

(Created page with "=Tuesday 19<sup>th</sup> July= ==Lab work== ===Biobrick characterization=== ====K1372001 from clone 2 digestion==== ''By Alice and Terrence'' We made a digestion of K1372001...")
 
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=Tuesday 19<sup>th</sup> July=
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{{Team:Paris_Saclay/notebook_header}}
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==Lab work==
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=Thursday 21<sup>st</sup> July=
  
 
===Biobrick characterization===
 
===Biobrick characterization===
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We made a digestion of K1372001 plasmid following this [[Team:Paris_Saclay/Experiments#PlasmidDigestion|protocol]].  
 
We made a digestion of K1372001 plasmid following this [[Team:Paris_Saclay/Experiments#PlasmidDigestion|protocol]].  
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We used EcoR1 and Pst1 fast digest enzymes.  
+
We used EcoRI and PstI fast digest enzymes.  
 
After incubation, 3.3 mL of loading dye is added to digestion products.  
 
After incubation, 3.3 mL of loading dye is added to digestion products.  
−
We put in wells 10µL of DNA ladder and 20 µL of digestion products.
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We put on gel 10µL of DNA ladder and 20 µL of digestion products.
 
We set the eletrophoresis machine at 100V for 25min.
 
We set the eletrophoresis machine at 100V for 25min.
  
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|-
 
|-
 
!Plasmid
 
!Plasmid
−
|
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|1590
 
|-
 
|-
 
!Insert
 
!Insert
−
|
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|2029
 
|}
 
|}
 +
[[File:T--Paris_Saclay--digestion_20160721_échelle.jpg|400px|thumb|right|Migration of digested product of K1372001]].
  
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====Making Petri dishes of medium LB (with streptomycin or IPTG and X-Gal)====
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''By Laetitia''
 +
 +
A Petri dish with LB and streptomycin was made with:
 +
* 20 mL of LB agar
 +
* 10µL of streptomycin (initial concentration 100mg/mL)
 +
 +
We added IPTG and xGal on Petri dishes of LB and Ampicillin.
 +
For each Petri dish:
 +
* 500µL of water
 +
* 1µL of IPTG
 +
* 1µL of X-Gal
 +
 +
The solution was spread on Petri dish
 +
 +
====Liquid culture of DH5a transformed with pcl_TAA, pcl_TAG and pcl_Tq====
 +
''By Laetitia''
 +
 +
2 clones of each Petri dish were inoculated in a tube of LB and Streptomycin.
 +
 +
6 tubes were made. For each tube:
 +
* 1 mL LB
 +
* 0.5 µL streptomycin
 +
 +
The tubes were incubated at 37°C and 180 rpm overnight.
  
 
===Visualization===
 
===Visualization===
−
==== ====
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====Low Fidelity Dreamtaq PCR of DH5α transformed with pPS16_002, pPS16_004 and pPS16_007 ====
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''By ''
+
''By Mathilde''
 +
 
 +
DH5α|pPS16_007 that were put in culture the day before showed blue colonies for the clones 5 and 11. Thus those clones can not be used for high fidelity PCR and be sequenced.
 +
 
 +
A DreamTaq PCR was made with transformed cultures  pPS16_002, pPS16_004 and pPS16_007 following the usual [[Team:Paris_Saclay/Experiments#Polymerase_chain_reaction|protocol]] with Tm at 57°C.
 +
 
 +
We divided up the PCR mix in 18 PCR tubes and added in each one a different clone from transformed each of our three cultures.
 +
The picked colonies were spread on a Petri dish medium LB + Ampicillin (50µg/mL) + X-Gal/IPTG (1/1000).
 +
 
 +
Results :
 +
PCR products expected were :
 +
{| class="wikitable"
 +
|-
 +
!'''Plasmid'''
 +
!pPS16_002
 +
!pPS16_004
 +
!pPS16_007
 +
|-
 +
!'''Bande Size (bp)'''
 +
|960
 +
|808
 +
|706
 +
|}
 +
 
 +
 
 +
[[File:T--Paris_Saclay--20160721_PCRpPS16_00247_échelle1.JPG|400px|thumb|right|]]
  
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===Get DNA Closer===
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The electropheresis on agarose gel showed good size product PCR for pPS16_004 clones 2 to 6, for pPS16_002 clone 5, and  pPS16_007 clones 2 and 3.
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==== ====
+
−
''By ''
+
  
 
{{Team:Paris_Saclay/notebook_footer}}
 
{{Team:Paris_Saclay/notebook_footer}}

Latest revision as of 15:54, 9 October 2016

Thursday 21st July

Biobrick characterization

K1372001 from clone 2 digestion

By Alice and Terrence

We made a digestion of K1372001 plasmid following this protocol. We used EcoRI and PstI fast digest enzymes. After incubation, 3.3 mL of loading dye is added to digestion products. We put on gel 10µL of DNA ladder and 20 µL of digestion products. We set the eletrophoresis machine at 100V for 25min.

Digestion products expected were :

Band size (Bp)
Plasmid 1590
Insert 2029
Migration of digested product of K1372001
.

Making Petri dishes of medium LB (with streptomycin or IPTG and X-Gal)

By Laetitia

A Petri dish with LB and streptomycin was made with:

  • 20 mL of LB agar
  • 10µL of streptomycin (initial concentration 100mg/mL)

We added IPTG and xGal on Petri dishes of LB and Ampicillin. For each Petri dish:

  • 500µL of water
  • 1µL of IPTG
  • 1µL of X-Gal

The solution was spread on Petri dish

Liquid culture of DH5a transformed with pcl_TAA, pcl_TAG and pcl_Tq

By Laetitia

2 clones of each Petri dish were inoculated in a tube of LB and Streptomycin.

6 tubes were made. For each tube:

  • 1 mL LB
  • 0.5 µL streptomycin

The tubes were incubated at 37°C and 180 rpm overnight.

Visualization

Low Fidelity Dreamtaq PCR of DH5α transformed with pPS16_002, pPS16_004 and pPS16_007

By Mathilde

DH5α|pPS16_007 that were put in culture the day before showed blue colonies for the clones 5 and 11. Thus those clones can not be used for high fidelity PCR and be sequenced.

A DreamTaq PCR was made with transformed cultures pPS16_002, pPS16_004 and pPS16_007 following the usual protocol with Tm at 57°C.

We divided up the PCR mix in 18 PCR tubes and added in each one a different clone from transformed each of our three cultures. The picked colonies were spread on a Petri dish medium LB + Ampicillin (50µg/mL) + X-Gal/IPTG (1/1000).

Results : PCR products expected were :

Plasmid pPS16_002 pPS16_004 pPS16_007
Bande Size (bp) 960 808 706


T--Paris Saclay--20160721 PCRpPS16 00247 échelle1.JPG

The electropheresis on agarose gel showed good size product PCR for pPS16_004 clones 2 to 6, for pPS16_002 clone 5, and pPS16_007 clones 2 and 3.





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