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

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{{Team:Paris_Saclay/notebook_header}}
 
=Thursday 13th August=
 
=Thursday 13th August=
 
==Lab Work==
 
==Lab Work==
Line 5: Line 6:
 
''by Pauline''
 
''by Pauline''
  
−
Biobricks:
 
 
* BBa_R0051 (2nd send)
 
* BBa_R0051 (2nd send)
 
* BBa_K1707011 #3 (x2)
 
* BBa_K1707011 #3 (x2)
Line 18: Line 18:
  
  
 +
===Digestion===
 +
''by Coralie''
  
 +
* 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===
 +
<html><i>by Pauline</i></html>
  
 +
Agarose gel, 1%, Migration 90V
  
 +
Biobricks:
 +
* Verification:
 +
** BBa_K1707016 #1 and #2
 +
** BBa_K1707031 #1 and #2
 +
* Purification:
 +
** BBa_K1707011 #3
 +
** BBa_K1707012 #2
 +
** BBa_K1707033 #1 and #2
 +
** BBa_R0051 (2<html><sup>nd</sup></html> send)
  
−
===Quantification===
+
[[File:ParisSaclay 13.08.15-vérif.jpg|300px|center]]
−
''by Pauline''
+
<html><p><i>Wells 2-5: Verification by digestion with XbaI and PstI, Wells 6-10: Verification of gel purification; from left to right: 1. <a href="https://2015.igem.org/File:Paris_Saclay-Ladder.jpg" target="_blank">DNA Ladder</a>, 2. BBa_K1707016#1, 3. BBa_K1707016#2, 4. BBa_K1707031#1, 5. BBa_K1707031#2,  6. BBa_K1707033#1, 7. BBa_K1707033#2, 8. BBa_K1707011#3, 9. BBa_K1707012#2, 10. BBa_R0051 (2<sup>nd</sup> send)</i></p></html>
 +
We can cut all purification bands.
 +
For the verification, we can say that BBa_K1707016 #1 and #2 are OK, but not BBa_K1707031 #1 or #2.
  
−
Agarose gel 1%, Migration 90V
+
We made a stock of BBa_K1707016 #1 and #2 and BBa_K1707033 #1 and #2.
  
−
We can conclude:
+
===Purification===
−
* 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''
 
'' by Pauline''
  
−
Agarose gel 1%, 90V
 
−
 
−
Biobricks:
 
 
* BBa_K1707011 #3
 
* BBa_K1707011 #3
 
* BBa_K1707012 #2
 
* BBa_K1707012 #2
 +
* BBa_K1707033 #1 and #2
 +
* BBa_R0051 (2nd send)
  
−
We can conclude that biobricks are OK
+
With Macherey-Nagel Purification kit
−
 
+
−
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===
 
===New culture===
−
''by Pauline''
+
'' 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
+
−
 
+
  
 +
* BBa_K1707031
 +
We put 4 new clones in 5mL LB + 5µL Chloramphenicol
  
 +
===Soil experiment===
 +
''by Coralie, Ibtissam, Johan and Claire''
  
 
'''Member present:'''
 
'''Member present:'''

Latest revision as of 20:35, 18 September 2015

Thursday 13th August

Lab Work

Plasmid extraction

by Pauline

  • 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

  • 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

by Pauline

Agarose gel, 1%, Migration 90V

Biobricks:

  • Verification:
    • BBa_K1707016 #1 and #2
    • BBa_K1707031 #1 and #2
  • Purification:
    • BBa_K1707011 #3
    • BBa_K1707012 #2
    • BBa_K1707033 #1 and #2
    • BBa_R0051 (2nd send)
ParisSaclay 13.08.15-vérif.jpg

Wells 2-5: Verification by digestion with XbaI and PstI, Wells 6-10: Verification of gel purification; from left to right: 1. DNA Ladder, 2. BBa_K1707016#1, 3. BBa_K1707016#2, 4. BBa_K1707031#1, 5. BBa_K1707031#2, 6. BBa_K1707033#1, 7. BBa_K1707033#2, 8. BBa_K1707011#3, 9. BBa_K1707012#2, 10. BBa_R0051 (2nd send)

We can cut all purification bands. For the verification, we can say that BBa_K1707016 #1 and #2 are OK, but not BBa_K1707031 #1 or #2.

We made a stock of BBa_K1707016 #1 and #2 and BBa_K1707033 #1 and #2.

Purification

by Pauline

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

With Macherey-Nagel Purification kit

New culture

by Pauline

  • BBa_K1707031

We put 4 new clones in 5mL LB + 5µL Chloramphenicol

Soil experiment

by Coralie, Ibtissam, Johan and Claire

Member present:

  • Instructors: Claire
  • Students: Coralie and Pauline

Back to the calendar

Contact
eigem.parissaclay AT gmail DOT com
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.