Difference between revisions of "Team:Paris Saclay/Notebook/August/13"

Line 18: Line 18:
  
  
 +
===Digestion===
 +
''by Coralie''
  
 +
Biobricks:
 +
* BBa_K1707011
 +
* BBa_K1707012
 +
* BBa_K1707016 #1 and #2
 +
* BBa_K1707031 #1 and #2
 +
* BBa_K1707033 #1 and #2
 +
* BBa_R0051
  
 +
Mixes:
 +
* BBa_K1707011 and BBa_K1707012
 +
** 1 µL XbaI
 +
** 2 µL PstI
 +
** 2 µL FastDigest Buffer 10x
 +
** 1 µL H2O
 +
** 15 µL plasmid
  
 +
* BBa_K1707016 #1 and #2 and BBa_K1707031 #1 and #2
 +
** 1 µL XbaI
 +
** 2 µL PstI
 +
** 2 µL FastDigest Buffer 10x
 +
** 11 µL H2O
 +
** 5 µL plasmid
 +
 +
* BBa_K1707033 #1 and #2
 +
** 1 µL XbaI
 +
** 2 µL PstI
 +
** 2 µL FastDigest Buffer 10x
 +
** 6 µL H2O
 +
** 10 µL plasmid
 +
 +
* BBa_R0051
 +
** 1 µL SpeI
 +
** 2 µL PstI
 +
** 2 µL Tango Buffer 10x
 +
** 5 µL H2O
 +
** 10 µL plasmid
 +
 +
Incubation 1h30, 37°C
 +
 +
 +
===Electrophoresis===
 +
  
  

Revision as of 20:31, 9 September 2015

Thursday 13th August

Lab Work

Plasmid extraction

by Pauline

Biobricks:

  • BBa_R0051 (2nd send)
  • BBa_K1707011 #3 (x2)
  • BBa_K1707012 #2 (x2)
  • BBa_K1707033 #1 and #2
  • BBa_K1707031 #1 and #2
  • BBa_K1707016 #1 and #2

With Macherey-Nagel Extraction kit (BBa_K1707011 #1 and #2 are assembled in the purification column) (BBa_K1707012 #1 and #2 are assembled in the purification column)


Digestion

by Coralie

Biobricks:

  • BBa_K1707011
  • BBa_K1707012
  • BBa_K1707016 #1 and #2
  • BBa_K1707031 #1 and #2
  • BBa_K1707033 #1 and #2
  • BBa_R0051

Mixes:

  • BBa_K1707011 and BBa_K1707012
    • 1 µL XbaI
    • 2 µL PstI
    • 2 µL FastDigest Buffer 10x
    • 1 µL H2O
    • 15 µL plasmid
  • BBa_K1707016 #1 and #2 and BBa_K1707031 #1 and #2
    • 1 µL XbaI
    • 2 µL PstI
    • 2 µL FastDigest Buffer 10x
    • 11 µL H2O
    • 5 µL plasmid
  • BBa_K1707033 #1 and #2
    • 1 µL XbaI
    • 2 µL PstI
    • 2 µL FastDigest Buffer 10x
    • 6 µL H2O
    • 10 µL plasmid
  • BBa_R0051
    • 1 µL SpeI
    • 2 µL PstI
    • 2 µL Tango Buffer 10x
    • 5 µL H2O
    • 10 µL plasmid

Incubation 1h30, 37°C


Electrophoresis

Quantification

by Pauline

Agarose gel 1%, Migration 90V

We can conclude:

  • BBa_I13602 #1: 40 µg/µL
  • BBa_I13602 #2: 40 µg/µL
  • BBa_K1707020: 10 µg/µL
  • BBa_K1707030: 13 µg/µL


Digestion

by Coralie

Biobricks:

  • BBa_K1707011 #3 and BBa_K1707012 #2
    • Mix for each reaction:
      • 1 µL PstI
      • 1 µL XbaI
      • 2 µL Buffer FastDigest 10x
      • 6 µL H2O
      • 10 µL plasmid
    • Incubation 37°C, 2h


Purification on gel

by Pauline

Agarose gel 1%, 90V

Biobricks:

  • BBa_K1707011 #3
  • BBa_K1707012 #2

We can conclude that biobricks are OK

Purification by the Macherey-Nagel Purification kit


Ligation

by Pauline

  • BBa_K1707035: BBa_K1707030 + BBa_I13602
    • 11,6 µL BBa_K1707030
    • 1,25 µL BBa_R0040
    • 2 µL Ligase
    • 2 µL Buffer 10x
    • 3,15 µL H2O
  • BBa_K1707036: BBa_K1707020 + BBa_I13602
    • 15,5 µL BBa_K1707006
    • 1,25 µL BBa_B0030
    • 2 µL Ligase
    • 2,5 µL Buffer 10x
    • 3,75 µL H2O


Incubation 4°C, ON


New culture

by Pauline

Biobricks:

  • From stock:
    • BBa_K1707012 #2 x2
    • BBa_K1707011 #3 x2
    • BBa_R0051
  • Two clones from plate of each:
    • BBa_K1707033
    • BBa_K1707031
    • BBa_K1707016

In 5mL LB + 5µL of appropriate antibiotic



Member present:

  • Instructors: Claire
  • Students: Coralie and Pauline

Back to the calendar

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.