Difference between revisions of "Team:Paris Saclay/Notebook/August/6"

(Created page with "=Thursday 6th August= ==Lab Work== ===Soil experiment=== ''by Coralie'' ====Water==== Observation of plates (05/08/2015) Almost all plates are yellow. We choose to stop the exp...")
 
m
 
(8 intermediate revisions by 4 users not shown)
Line 1: Line 1:
 +
{{Team:Paris_Saclay/notebook_header}}
 
=Thursday 6th August=
 
=Thursday 6th August=
 
==Lab Work==
 
==Lab Work==
Line 39: Line 40:
  
  
 +
===Purification agarose gel===
 +
''by Pauline and Audrey''
  
 +
* BBa_K1707012
 +
* BBa_K1707004
 +
* BBa_K1707011
 +
* BBa_R0051
 +
* BBa_K1707008
 +
* BBa_I13602
 +
* BBa_I13600
  
 +
And PIJ773 digested by NotI
  
−
===Quantification===
+
Agarose gel 1%
−
''by Pauline and Coralie''
+
  
−
Biobricks:
+
Migration 90 V
−
* BBa_R0051
+
[[File:ParisSaclay 06.08.15-Purification.jpg|400px|center]]
−
* BBa_E0022 (15ng/mL)
+
<html>
−
* BBa_E0422 (20ng/mL)
+
<p><i>Verification of gel purification, from left to right: 1. BBa_PIJ773 2. <a href="https://2015.igem.org/File:Paris_Saclay-Ladder.jpg" target="_blank">DNA Ladder</a>, 3. BBa_K1707004, 4. BBa_K1707008, 5. BBa_K1707011, 6. BBa_K1707012, 7. BBa_R0051, 8. BBa_I13600, 9. BBa_I13602, 10. Empty</i></p></html>
−
* BBa_K1707000 (20ng/mL)
+
We made a mistake: BBa_K1707008, BBa_I13602 and Bba_R0051 shouldn't be purified on agarose gel.
−
* BBa_K1707004 (15ng/mL)
+
We can observe a big smear for PIJ773.
−
* BBa_K1707011
+
We can also observe that BBa_I13602 has three bands.
−
* BBa_K1707012
+
Rest is alright.
−
* BBa_K1707013 (20ng/mL)
+
−
* BBa_K1707019 (15ng/mL)
+
  
−
Agarose gel, 1%
+
===Purification===
−
Migration 90V
+
−
 
+
−
 
+
−
===Ligation===
+
 
''by Pauline''
 
''by Pauline''
  
−
Biobricks:
+
* BBa_K1707011 #1
−
BBa_K1707020 : BBa_K1707004 + BBa_K1707019
+
* BBa_K1707004 #1
 +
* BBa_K1707012 #1
 +
* BBa_R0051
 +
* BBa_K1707008 #1
 +
* BBa_I136000
 +
* BBa_I13602
  
−
Mix:
+
With Macherey-Nagel Kit
−
* 6,5 µL BBa_K1707019
+
−
* 9,5 µL BBa_K1707004
+
−
* 2 µL Tampon 10x
+
−
* 1 µL Ligase
+
−
* 1 µL H2O
+
  
  
 +
===Quantification===
 +
'' by Pauline and Audrey''
  
−
BBa Test : BBa_K1707004 + BBa_K1707013
+
* BBa_K1707011 #1
 +
* BBa_K1707004 #1
 +
* BBa_K1707012 #1
 +
* BBa_R0051
 +
* BBa_K1707008 #1
 +
* BBa_I136000
 +
* BBa_I13602
  
−
Mix:
+
PIJ773 digested by NotI
−
* 5 µL BBa_K1707013
+
−
* 7,5 µL BBa_K1707004
+
−
* 1,5 µL Tampon 10x
+
−
* 1 µL Ligase
+
  
 +
Agarose gel, 1%
  
 +
Migration 100V
 +
[[File:ParisSaclay 06.08.15-Purif.jpg|400px|center]]
 +
<html><p><i>Wells 2-8: Quantification, Wells 9-10: Verification; from left to right: 1. <a href="https://2015.igem.org/File:Paris_Saclay-Ladder.jpg" target="_blank">DNA Ladder</a>, 2. BBa_K1707012#1, 3. BBa_I13602, 4. BBa_I13600, 5. BBa_K1707011#1, 6. BBa_K1707004#1, 7. BBa_K1707008#1, 8. BBa_R0051, 9. BBa_PIJ773, 10. PIJ773 plasmid</i></p></html>
  
 +
===Ligation===
 +
''by Audrey''
  
−
BBa_K1707027 : BBa_K1707000 + BBa_E0022
+
BBa_K1707026:
 +
* BBa_E0422 + BBa_J23101
  
 
Mix:
 
Mix:
−
* 5 µL BBa_K1707000
+
* 11 µL BBa_E0422
−
* 9 µL BBa_E0022
+
* 8 µL BBa_J23101
−
* 1,5 µL Tampon 10x
+
* 1,5 µL Buffer 10x
−
* 1 µL Ligase
+
* 2 µL Ligase
−
* 3,5 µL H2O
+
* 2,5 µL H2O
  
 +
BBa_K1707032:
 +
* BBa_I13600 + BBa_K1707008
  
 +
Mix:
 +
* 2 µL BBa_K1707008
 +
* 5 µL BBa_I13600
 +
* 1 µL Buffer 10x
 +
* 2 µL Ligase
  
−
 
+
BBa_K1707033:
−
BBa_K1707028 : BBa_R0051 + BBa_E0422
+
* BBa_J23101 + BBa_K1707006
  
 
Mix:
 
Mix:
−
* 6,25 µL BBa_R0051
+
* 6,5 µL BBa_K1707006
−
* 5,5 µL BBa_E0422
+
* 1,5 µL BBa_J23101
 
* 1,5 µL Tampon 10x
 
* 1,5 µL Tampon 10x
−
* 1 µL Ligase
+
* 2 µL Ligase
−
* 0,75 µL H2O
+
* 3,5 µL H2O
  
−
===Rehydratation===
+
Incubation ON, 4°C
−
''by Audrey''
+
  
−
BBa_R0040
+
===New culture===
−
 
+
''by Pauline''
−
===Transformation===
+
−
''by Audrey and Coralie''
+
  
 +
* BBa_K1707020
 +
* Plamsid test
 +
* BBa_K1707027
 
* BBa_R0040
 
* BBa_R0040
−
* BBa Test
 
 
* BBa_K1707028
 
* BBa_K1707028
−
* BBa_K1707027
 
−
* BBa_K1707020
 
−
 
−
===Digestion===
 
−
''by Pauline''
 
−
 
−
XbaI + PstI:
 
−
* BBa_K1707012
 
−
* BBa_K1707011
 
−
* BBa_I13600
 
−
 
−
SpeI + Pst:
 
−
* BBa_R0051
 
−
* BBa_K1707008
 
−
 
−
EcoRI + SpeI:
 
−
* BBa_K1707004
 
−
 
−
XbaI + EcoRI:
 
−
* BBa_I13602
 
−
 
−
 
−
Mix:
 
−
* 10 µL Plasmide
 
−
* 1 µL Enzyme 1
 
−
* 1 µL Enzyme 2
 
−
* 2 µL Buffer FastDigest 10x
 
−
* 6 µL H2O
 
−
 
−
 
  
 +
2clones in 5mL LB + antibiotics
  
 
'''Member present:'''
 
'''Member present:'''

Latest revision as of 22:10, 18 September 2015

Thursday 6th August

Lab Work

Soil experiment

by Coralie

Water

Observation of plates (05/08/2015) Almost all plates are yellow. We choose to stop the experiment.

We will define a new protocol:

  • Count of bacteria before contamination
  • Evaluation of the count of bacteria we should see at Day0
  • Measurement of the pH before each sampling (We think that maybe the water pH change with adding the LB)

Low Budget Challenge

by Coralie

We can't say a lot of things... But we can see some colony on the agar with the baker's yeast in the agar. We try another time with different agar recipes:

  • Agar 1:
    • 0,27g Stock Cube
    • 0,600g Agar Agar
    • 0,135g Baker's yeast
    • 0,225g sugar
    • 45mL mineral water
  • Agar 2:
    • 0,27g Stock Cube
    • 0,600g Agar Agar
    • 0,135g Baker's yeast
    • 45mL mineral water

We make striations with BBa_J23101+BBa_I13504 strain with a curved pin. We do the same on an usual LB plate without antibiotics (Control +)

Incubation over night, 37°C


Purification agarose gel

by Pauline and Audrey

  • BBa_K1707012
  • BBa_K1707004
  • BBa_K1707011
  • BBa_R0051
  • BBa_K1707008
  • BBa_I13602
  • BBa_I13600

And PIJ773 digested by NotI

Agarose gel 1%

Migration 90 V

ParisSaclay 06.08.15-Purification.jpg

Verification of gel purification, from left to right: 1. BBa_PIJ773 2. DNA Ladder, 3. BBa_K1707004, 4. BBa_K1707008, 5. BBa_K1707011, 6. BBa_K1707012, 7. BBa_R0051, 8. BBa_I13600, 9. BBa_I13602, 10. Empty

We made a mistake: BBa_K1707008, BBa_I13602 and Bba_R0051 shouldn't be purified on agarose gel. We can observe a big smear for PIJ773. We can also observe that BBa_I13602 has three bands. Rest is alright.

Purification

by Pauline

  • BBa_K1707011 #1
  • BBa_K1707004 #1
  • BBa_K1707012 #1
  • BBa_R0051
  • BBa_K1707008 #1
  • BBa_I136000
  • BBa_I13602

With Macherey-Nagel Kit


Quantification

by Pauline and Audrey

  • BBa_K1707011 #1
  • BBa_K1707004 #1
  • BBa_K1707012 #1
  • BBa_R0051
  • BBa_K1707008 #1
  • BBa_I136000
  • BBa_I13602

PIJ773 digested by NotI

Agarose gel, 1%

Migration 100V

ParisSaclay 06.08.15-Purif.jpg

Wells 2-8: Quantification, Wells 9-10: Verification; from left to right: 1. DNA Ladder, 2. BBa_K1707012#1, 3. BBa_I13602, 4. BBa_I13600, 5. BBa_K1707011#1, 6. BBa_K1707004#1, 7. BBa_K1707008#1, 8. BBa_R0051, 9. BBa_PIJ773, 10. PIJ773 plasmid

Ligation

by Audrey

BBa_K1707026:

  • BBa_E0422 + BBa_J23101

Mix:

  • 11 µL BBa_E0422
  • 8 µL BBa_J23101
  • 1,5 µL Buffer 10x
  • 2 µL Ligase
  • 2,5 µL H2O

BBa_K1707032:

  • BBa_I13600 + BBa_K1707008

Mix:

  • 2 µL BBa_K1707008
  • 5 µL BBa_I13600
  • 1 µL Buffer 10x
  • 2 µL Ligase

BBa_K1707033:

  • BBa_J23101 + BBa_K1707006

Mix:

  • 6,5 µL BBa_K1707006
  • 1,5 µL BBa_J23101
  • 1,5 µL Tampon 10x
  • 2 µL Ligase
  • 3,5 µL H2O

Incubation ON, 4°C

New culture

by Pauline

  • BBa_K1707020
  • Plamsid test
  • BBa_K1707027
  • BBa_R0040
  • BBa_K1707028

2clones in 5mL LB + antibiotics

Member present:

  • Instructors: Claire
  • Students: Coralie, Audrey 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.