Difference between revisions of "Team:Paris Saclay/Notebook/August/11"

(Created page with "=Tuesday 11th August= ==Lab Work== ===Ligation=== ''by Pauline'' * BBa_K1707031: BBa_K1707004 + BBa_R0040 ** 10,4 µL BBa_K1707004 digested by EcoRI + SpeI ** 3,3 µL BBa_R0040...")
 
Line 13: Line 13:
  
 
* BBa_K1707033: BBa_K1707006 + BBa_B0030
 
* BBa_K1707033: BBa_K1707006 + BBa_B0030
−
** 7,3 µL BBa_K1707006 digested by EcoRI + SpeI
+
** 7,3 µL BBa_K1707006 digested by XbaII + PstI
 
** 7,4 µL BBa_B0030  
 
** 7,4 µL BBa_B0030  
 
** 2 µL Ligase
 
** 2 µL Ligase
Line 19: Line 19:
 
** 5,8 µL H2O
 
** 5,8 µL H2O
  
 +
* BBa_K1707016: BBa_K1707004 + BBa_I13602
 +
** 7,2 µL BBa_K1707004 digested by EcoRI + SpeI
 +
** 2 µL BBa_B0030
 +
** 2 µL Ligase
 +
** 1,5 µL Buffer 10x
 +
** 2,5 µL H2O
 +
 +
Incubation 4°C, 3h
 +
 +
===Plamid Extraction===
 +
''by Pauline''
 +
 +
Biobricks:
 +
* BBa_K1707026 #1 and #2
 +
* BBa_K1707032 #1 and #2
 +
 +
With Macherey-Nagel Extraction kit
 +
 +
 +
===Digestion===
 +
''by Coralie''
 +
 +
Biobricks:
 +
* BBa_K1707026 #1 and #2
 +
* BBa_K1707032 #1 and #2
 +
 +
Mix for each reaction:
 +
* 0,5 µL PstI
 +
* 0,5 µL XbaI
 +
* 1 µL Buffer FastDigest 10x
 +
* 6 µL H2O
 +
* 2 µL plasmid
 +
 +
Incubation 37°C, 2h
 +
 +
 +
===Electrophoresis===
 +
''by Pauline''
 +
 +
Agarose gel 1%, migration 90V
 +
 +
Biobricks:
 +
* Quantification:
 +
** BBa_K1707011 #3 (plasmid)
 +
** BBa_K1707012 #2 (plasmid)
 +
* Verification of digestion:
 +
** BBa_K1707026 #1
 +
** BBa_K1707026 #2
 +
** BBa_K1707028 #1
 +
** BBa_K1707028 #2
 +
** BBa_K1707032 #1
 +
** BBa_K1707032 #2
 +
 +
We can conclude:
 +
* Quantification:
 +
** BBa_K1707011 #3 (plasmid): 13 µg/µL
 +
** BBa_K1707012 #2 (plasmid): 20 µg/µL
 +
* Verification of digestion:
 +
** BBa_K1707026 #1: OK but not totally digested
 +
** BBa_K1707026 #2: OK but not totally digested
 +
** BBa_K1707028 #1: OK
 +
** BBa_K1707028 #2: OK
 +
** BBa_K1707032 #1: OK but not totally digested
 +
** BBa_K1707032 #2: OK but not totally digested
 +
 +
 +
===Digestion===
 +
''by Coralie''
 +
 +
Biobricks:
 +
* BBa_I13602 x2
 +
** Mix for each reaction:
 +
*** 1 µL EcoRI
 +
*** 1 µL XbaI
 +
*** 2 µL Buffer FastDigest 10x
 +
*** 6 µL H2O
 +
*** 10 µL plasmid
 +
** Incubation 37°C, 2h
 +
 +
* BBa_K1707030 and BBa_K1707020
 +
** Mix for each reaction:
 +
*** 2 µL Tango Buffer
 +
*** 1 µL SpeI
 +
*** 7 µL H2O
 +
*** 10 µL Plasmid
 +
** Incubation 2h, 37°C
 +
** After incubation: we add in each tube:
 +
*** 3 µL Tango Buffer
 +
*** 1 µL EcoRI
 +
*** 1 µL H2O
 +
** Incubation 1h30, 37°C
 +
 +
 +
===Purification on gel===
 +
'' by Pauline''
  
 +
Biobricks:
 +
* BBa_K1707020
 +
* BBa_K1707030
  
 +
We can conclude that biobricks are OK
  
  
 +
===Transformation===
 +
''by Pauline''
  
 +
Biobricks:
 +
* BBa_K1707016
 +
* BBa_K1707031
 +
* BBa_K1707033
  
 +
As usual
  
 +
===Low cost experiment===
 +
''by Coralie''
  
 +
We make 8 plates of home
  
  

Revision as of 18:03, 9 September 2015

Tuesday 11th August

Lab Work

Ligation

by Pauline

  • BBa_K1707031: BBa_K1707004 + BBa_R0040
    • 10,4 µL BBa_K1707004 digested by EcoRI + SpeI
    • 3,3 µL BBa_R0040 digested by EcoRI + XbaI
    • 2 µL Ligase
    • 2 µL Buffer 10x
    • 2,3 µL H2O
  • BBa_K1707033: BBa_K1707006 + BBa_B0030
    • 7,3 µL BBa_K1707006 digested by XbaII + PstI
    • 7,4 µL BBa_B0030
    • 2 µL Ligase
    • 2,5 µL Buffer 10x
    • 5,8 µL H2O
  • BBa_K1707016: BBa_K1707004 + BBa_I13602
    • 7,2 µL BBa_K1707004 digested by EcoRI + SpeI
    • 2 µL BBa_B0030
    • 2 µL Ligase
    • 1,5 µL Buffer 10x
    • 2,5 µL H2O

Incubation 4°C, 3h

Plamid Extraction

by Pauline

Biobricks:

  • BBa_K1707026 #1 and #2
  • BBa_K1707032 #1 and #2

With Macherey-Nagel Extraction kit


Digestion

by Coralie

Biobricks:

  • BBa_K1707026 #1 and #2
  • BBa_K1707032 #1 and #2

Mix for each reaction:

  • 0,5 µL PstI
  • 0,5 µL XbaI
  • 1 µL Buffer FastDigest 10x
  • 6 µL H2O
  • 2 µL plasmid

Incubation 37°C, 2h


Electrophoresis

by Pauline

Agarose gel 1%, migration 90V

Biobricks:

  • Quantification:
    • BBa_K1707011 #3 (plasmid)
    • BBa_K1707012 #2 (plasmid)
  • Verification of digestion:
    • BBa_K1707026 #1
    • BBa_K1707026 #2
    • BBa_K1707028 #1
    • BBa_K1707028 #2
    • BBa_K1707032 #1
    • BBa_K1707032 #2

We can conclude:

  • Quantification:
    • BBa_K1707011 #3 (plasmid): 13 µg/µL
    • BBa_K1707012 #2 (plasmid): 20 µg/µL
  • Verification of digestion:
    • BBa_K1707026 #1: OK but not totally digested
    • BBa_K1707026 #2: OK but not totally digested
    • BBa_K1707028 #1: OK
    • BBa_K1707028 #2: OK
    • BBa_K1707032 #1: OK but not totally digested
    • BBa_K1707032 #2: OK but not totally digested


Digestion

by Coralie

Biobricks:

  • BBa_I13602 x2
    • Mix for each reaction:
      • 1 µL EcoRI
      • 1 µL XbaI
      • 2 µL Buffer FastDigest 10x
      • 6 µL H2O
      • 10 µL plasmid
    • Incubation 37°C, 2h
  • BBa_K1707030 and BBa_K1707020
    • Mix for each reaction:
      • 2 µL Tango Buffer
      • 1 µL SpeI
      • 7 µL H2O
      • 10 µL Plasmid
    • Incubation 2h, 37°C
    • After incubation: we add in each tube:
      • 3 µL Tango Buffer
      • 1 µL EcoRI
      • 1 µL H2O
    • Incubation 1h30, 37°C


Purification on gel

by Pauline

Biobricks:

  • BBa_K1707020
  • BBa_K1707030

We can conclude that biobricks are OK


Transformation

by Pauline

Biobricks:

  • BBa_K1707016
  • BBa_K1707031
  • BBa_K1707033

As usual

Low cost experiment

by Coralie

We make 8 plates of home




Digestion verification

by Coralie

Biobricks:

  • BBa_K1707004 #2
  • BBa_K1707011 #1
  • BBa_ROO407#1
  • BBa_K1707006 #1
  • BBa_I13602 #1
  • BBa_K1707012 #1
  • BBa_R0051 #1
  • BBa_S03518 #1


  • Mix for BBa_S03518 and BBa_K1707006:
    • 10 µL plasmid
    • 1 µL XbaI
    • 1 µL PstI
    • 2 µL Buffer FastDigest 10x
    • 6 µL H2O


  • Mix for BBa_K1707011 and BBa_K1707012:
    • 15 µL plasmid
    • 1 µL XbaI
    • 1 µL PstI
    • 2 µL Buffer FastDigest 10x
    • 1 µL H2O


  • Mix for BBa_R0040 and BBa_I13602:
    • 10 µL plasmid
    • 1 µL EcoRI
    • 1 µL XbaI
    • 2 µL Buffer FastDigest 10x
    • 6 µL H2O


  • Mix for BBa_R0051
    • 10 µL plasmid
    • 1 µL SpeI
    • 2 µL PstI
    • 2 µL Tango Buffer 10x
    • 6 µL H2O

Incubation 1h30, 37°C


  • Mix for BBa_K1707004:
    • 2 µL Tango Buffer 10x
    • 1 µL SpeI
    • 2 µL H2O
    • 15 µL plasmid

Incubation 1h30, 37°C After incubation, we add:

    • 3 µL Tango Buffer 10x
    • 1 µL EcoRI
    • 1 µL H2O

Incubation 1h30, 37°C


Purification by Electrophoresis

by Coralie

Agarose gel 1%, 100V

Biobricks:

  • BBa_K1707004 #1 and #2 (digested by EcoRI + SpeI)
  • BBa_K1707011 #1 (digested by XbaI +PstI)
  • BBa_K1707006 #1 (digested by XbaI + PstI)
  • BBa_K1707012 #1 (digested by XbaI + PstI)
  • BBa_S03518 #1 (digested by XbaI + PstI)


We can conclude that:

  • BBa_K1707004 #1 and #2 are OK, we can cut the band
  • BBa_K1707006 is OK, we can cut the band
  • BBa_K1707011: we can't see anything on the gel
  • BBa_K1707012: we can't see anything on the gel
  • BBa_S03518: is OK, we can cut the band


Purification

by Pauline

Biobricks:

  • BBa_K1707004 #2
  • BBa_K1707011 #1
  • BBa_K1707006 #1
  • BBa_K1707012 #1
  • BBa_S03518 #1
  • BBa_R0040 #1
  • BBa_I13602 #1
  • BBa_R0051 #1

With Macherey-Nagel Purification kit


Quantification

by Pauline

Biobricks:

  • BBa_K1707004 #2 x2
  • BBa_K1707006 #1
  • BBa_S03518 #1
  • BBa_R0040 #1
  • BBa_I13602 #1
  • BBa_R0051 #1

Agarose gel 1%, migration at 100V

We can conclude:

  • BBa_K1707004 #2: 20µg/µL
  • BBa_K1707006 #1: 30µg/µL
  • BBa_S03518 #1: 50µg/µL
  • BBa_R0040 #1: 30 µg/µL
  • BBa_I13602 #1: 50µg/µL
  • BBa_R0051 #1: we can't see anything


New Culture

by Pauline

Biobricks:

  • BBa_K1707026
  • BBa_K1707032

We put two clones of each in 5mL LB + 5µL Chloramphenicol


Member present:

  • Instructors: Claire
  • Students: Coralie and Pauline

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Contact
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t@iGEMParisSaclay
aUniversité Paris Sud
a91405 Orsay, France Copyright © 2015 iGEM Paris Saclay. All rights reserved All inspirations encouraged.
*This wiki was conceived during long nights of work and a great coffee experience in the beautiful Orsay, France.