Difference between revisions of "Team:Paris Saclay/Notebook/August/14"

(Created page with "=Friday 14th August= ==Lab Work== ===Quantification=== ''by Pauline'' Biobricks: * BBa_R0051 (2nd send) * BBa_K1707011 #3 * BBa_K1707012 #2 * BBa_K1707033 #1 and #2 Mix: * 2...")
 
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{{Team:Paris_Saclay/notebook_header}}
 
=Friday 14th August=
 
=Friday 14th August=
 
==Lab Work==
 
==Lab Work==
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''by Pauline''
 
''by Pauline''
  
−
Biobricks:
 
 
* BBa_R0051 (2nd send)
 
* BBa_R0051 (2nd send)
 
* BBa_K1707011 #3  
 
* BBa_K1707011 #3  
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Agarose gel 1%, Migration 90V
 
Agarose gel 1%, Migration 90V
−
 
+
[[File:ParisSaclay 14.08.15-quantif(3).jpg|300px|center]]
 +
<html><p><i>Quantification, from left to right: 1. <a href="https://2015.igem.org/File:Paris_Saclay-Ladder.jpg" target="_blank">DNA Ladder</a>, 2. BBa_K1707011#3 (08/12), 3. BBa_K1707011#3 (08/12), 4. BBa_K1707012#2 (08/12), 5. BBa_K1707012#2 (08/13),  6. BBa_K1707033#1, 7. BBa_K1707033#2, 8. BBa_R0051 (2<sup>nd</nd> send), 9. Empty, 10. Empty</i></p></html>
 
We can conclude:
 
We can conclude:
 
* BBa_R0051 (2nd send): 13 µg/µL  
 
* BBa_R0051 (2nd send): 13 µg/µL  
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'' by Pauline''
 
'' by Pauline''
  
−
Biobricks:
 
 
* BBa_K1707035
 
* BBa_K1707035
 
* BBa_K1707036
 
* BBa_K1707036
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''by Pauline''
 
''by Pauline''
  
−
BIobricks:
 
 
* BBa_K1707031 #3, #4, #5 and #6
 
* BBa_K1707031 #3, #4, #5 and #6
  
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''by Pauline''
 
''by Pauline''
  
−
Biobricks:
 
 
* BBa_K1707031 #3, #4, #5 and #6
 
* BBa_K1707031 #3, #4, #5 and #6
  
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Incubation 37°C, 1H30
 
Incubation 37°C, 1H30
−
 
  
 
===Ligation===
 
===Ligation===
 
''by Pauline''
 
''by Pauline''
  
−
* BBa_K1707023: BBa_K1707012 and BBa_K1707031 #1 and #2
+
* BBa_K1707023: BBa_K1707012 and BBa_J23101
−
** 1 µL XbaI
+
** 5,9 µL BBa_K1707012
−
** 2 µL PstI
+
** 5 µL BBa_J23101
−
** 2 µL FastDigest Buffer 10x
+
** 2 µL Ligase
−
** 11 µL H2O
+
** 1,5 µL Buffer
−
** 5 µL plasmid
+
** 0,6 µL H2O
  
−
* BBa_K1707033 #1 and #2
+
* BBa_K1707022: BBa_K1707011 and BBa_R0051
−
** 1 µL XbaI
+
** 5,4 µL BBa_K1707011
−
** 2 µL PstI
+
** 7,6 µL BBa_R0051
−
** 2 µL FastDigest Buffer 10x
+
** 2 µL Ligase
−
** 6 µL H2O
+
** 2 µL Buffer  
−
** 10 µL plasmid
+
** 3 µL H2O
−
 
+
−
* BBa_R0051
+
−
** 1 µL SpeI
+
−
** 2 µL PstI
+
−
** 2 µL Tango Buffer 10x
+
−
** 5 µL H2O
+
−
** 10 µL plasmid
+
  
−
Incubation 1h30, 37°C
+
* BBa_K1707034: BBa_K1707033 and BBa_J23101
 +
** 11 µL BBa_K1707033
 +
** 5 µL BBa_J23101
 +
** 2 µL Ligase
 +
** 2 µL Buffer
  
 +
Incubation 3h, 4°C
  
 
===Electrophoresis===
 
===Electrophoresis===
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Agarose gel, 1%, Migration 90V
 
Agarose gel, 1%, Migration 90V
−
 
+
[[File:ParisSaclay 14.08.15-vérif.jpg|300px|center]]
 +
<html><p><i>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_K1707030#3, 3. BBa_K1707031#4, 4. BBa_K1707031#5, 5. BBa_K1707031#6,  6. Empty, 7. Empty, 8. Empty, 9. Empty, 10. Empty</i></p></html>
 
Biobricks:
 
Biobricks:
 
* Verification:  
 
* Verification:  
−
** BBa_K1707016 #1 and #2
+
** BBa_K1707031 #3, #4, #5 and #6
−
** BBa_K1707031 #1 and #2
+
−
* Purification:
+
−
** BBa_K1707011 #3
+
−
** BBa_K1707012 #2
+
−
** BBa_K1707033 #1 and #2
+
−
** BBa_R0051 (2nd send)
+
  
−
We can cut all purification bands.
+
We can conclude that all clones aren't OK because the band viewed corresponding to the BBa_R0040 plasmid
−
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.
+
===Transformation===
−
 
+
''by Pauline''
−
 
+
−
===Purification===
+
−
'' by Pauline''
+
−
 
+
−
Biobricks:
+
−
* BBa_K1707011 #3
+
−
* BBa_K1707012 #2
+
−
* BBa_K1707033 #1 and #2
+
−
* BBa_R0051 (2nd send)
+
−
 
+
−
With Macherey-Nagel Purification kit
+
−
 
+
−
 
+
−
===New culture===
+
−
'' by Pauline''
+
−
 
+
−
Biobrick: BBa-K1707031
+
−
We put 4 new clones in 5mL LB + 5µL Chloramphenicol
+
  
 +
* BBa_K1707022
 +
* BBa_K1707023
 +
* BBa_K1707034
  
 
===Soil experiment===
 
===Soil experiment===
−
''by Coralie, Ibtissam, Johan and Claire''
+
''by Coralie and Johan''
−
 
+
−
 
+
  
 +
===Low budget experiment===
 +
''by Coralie''
  
 
'''Member present:'''
 
'''Member present:'''

Latest revision as of 20:49, 18 September 2015

Friday 14th August

Lab Work

Quantification

by Pauline

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

Mix:

  • 2 µL plasmid
  • 2 µL DNA Loading 6X
  • 6 µL H2O

Agarose gel 1%, Migration 90V

ParisSaclay 14.08.15-quantif(3).jpg

Quantification, from left to right: 1. DNA Ladder, 2. BBa_K1707011#3 (08/12), 3. BBa_K1707011#3 (08/12), 4. BBa_K1707012#2 (08/12), 5. BBa_K1707012#2 (08/13), 6. BBa_K1707033#1, 7. BBa_K1707033#2, 8. BBa_R0051 (2nd send), 9. Empty, 10. Empty

We can conclude:

  • BBa_R0051 (2nd send): 13 µg/µL
  • BBa_K1707011 #3: 40 µg/µL
  • BBa_K1707012 #2: 35 µg/µL
  • BBa_K1707033 #1 and #2: 20 µg/µL


New culture

by Pauline

  • BBa_K1707035
  • BBa_K1707036

We put 2 new clones in 5mL LB + 5µL Chloramphenicol


Plasmid extraction

by Pauline

  • BBa_K1707031 #3, #4, #5 and #6

With Macherey-Nagel Extraction kit


Digestion

by Pauline

  • BBa_K1707031 #3, #4, #5 and #6

Mix for each:

  • BBa_K1707011 and BBa_K1707012
    • 0,5 µL XbaI
    • 0,5 µL PstI
    • 1 µL FastDigest Buffer 10x
    • 6 µL H2O
    • 2 µL plasmid

Incubation 37°C, 1H30

Ligation

by Pauline

  • BBa_K1707023: BBa_K1707012 and BBa_J23101
    • 5,9 µL BBa_K1707012
    • 5 µL BBa_J23101
    • 2 µL Ligase
    • 1,5 µL Buffer
    • 0,6 µL H2O
  • BBa_K1707022: BBa_K1707011 and BBa_R0051
    • 5,4 µL BBa_K1707011
    • 7,6 µL BBa_R0051
    • 2 µL Ligase
    • 2 µL Buffer
    • 3 µL H2O
  • BBa_K1707034: BBa_K1707033 and BBa_J23101
    • 11 µL BBa_K1707033
    • 5 µL BBa_J23101
    • 2 µL Ligase
    • 2 µL Buffer

Incubation 3h, 4°C

Electrophoresis

by Pauline

Agarose gel, 1%, Migration 90V

ParisSaclay 14.08.15-vérif.jpg

Verification of gel purification, from left to right: 1. DNA Ladder, 2. BBa_K1707030#3, 3. BBa_K1707031#4, 4. BBa_K1707031#5, 5. BBa_K1707031#6, 6. Empty, 7. Empty, 8. Empty, 9. Empty, 10. Empty

Biobricks:

  • Verification:
    • BBa_K1707031 #3, #4, #5 and #6

We can conclude that all clones aren't OK because the band viewed corresponding to the BBa_R0040 plasmid

Transformation

by Pauline

  • BBa_K1707022
  • BBa_K1707023
  • BBa_K1707034

Soil experiment

by Coralie and Johan

Low budget experiment

by Coralie

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.