Difference between revisions of "Team:Paris Saclay/Notebook/August/4"

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{{Team:Paris_Saclay/notebook_header}}
 
=Tuesday 4th August=
 
=Tuesday 4th August=
 
==Lab Work==
 
==Lab Work==
Line 4: Line 5:
 
''by Pauline''
 
''by Pauline''
  
−
Biobricks:
 
 
* BBa_K1707008 #1 to #6
 
* BBa_K1707008 #1 to #6
 
* BBa_I13600 #1 and #2
 
* BBa_I13600 #1 and #2
Line 13: Line 13:
 
''by Pauline and Coralie''
 
''by Pauline and Coralie''
  
−
Biobricks: BBa_K1707008 #1 to #6
+
* BBa_K1707008 #1 to #6
  
 
Mix:
 
Mix:
Line 30: Line 30:
 
Agarose gel 1%
 
Agarose gel 1%
 
Migration 90V
 
Migration 90V
−
 
+
[[File:ParisSaclay 04.08.15-Digestion vérif 008.jpg|300px|center]]
 +
<html><i><p>Verification by digestion with XbaI and PstI of BBa_K1707008, from left to right: 1. <a href="https://2015.igem.org/File:Paris_Saclay-Ladder.jpg" target="_blank">DNA Ladder</a>, 2. #1, 3. #2, 4. #3, 5. #4, 6. #5, 7. #6, 8. Empty, 9. Empty, 10. Empty, 11. Empty, 12. Empty</p></i></html>
 
We can conclude that all BBa_K1707008 clones are OK
 
We can conclude that all BBa_K1707008 clones are OK
 +
 
We make a glycerol stock of #1 and #2
 
We make a glycerol stock of #1 and #2
  
  
−
===Electrophoresis===
+
===Electrophoresis Verification===
 
''by Pauline and Coralie''
 
''by Pauline and Coralie''
  
Line 41: Line 43:
  
 
Migration 100V
 
Migration 100V
−
 
+
[[File:Paris Saclay-03.08.2015 - digestion verification 2.jpg|400px|center]]
 +
<html>
 +
<i>Verification, from left to right: 1. <a href="https://2015.igem.org/File:Paris_Saclay-Ladder.jpg" target="_blank">DNA Ladder</a>, 2. BBa_K1707021#1, 3. BBa_K1707021#2, 4. BBa_K1707021#3, 5. BBa_K1707021#4, 6. BBa_K1707021#5, 7. BBa_K1707021#6, 8. BBa_K1707011#4, 9. BBa_K1707011#5, 10. BBa_K1707011#6</i></html>
 +
[[File:Paris Saclay-03.08.15 - digestion verification 1.jpg|400px|center]]
 +
<html>
 +
<p><i>Verification, from left to right: 1. <a href="https://2015.igem.org/File:Paris_Saclay-Ladder.jpg" target="_blank">DNA Ladder</a>, 2. BBa_K1707004#1, 3. BBa_K1707004#2, 4. BBa_K1707004#3, 5. BBa_K1707004#4, 6. BBa_K1707004#5, 7. BBa_K1707004#6, 8. BBa_K1707011#1, 9. BBa_K1707011#2, 10. BBa_K1707011#3, 11. Empty, 12. Empty</i></p></html>
 
We can conclude that:
 
We can conclude that:
 
* BBa_K1707021: #2, #3, #4 and #6 are OK
 
* BBa_K1707021: #2, #3, #4 and #6 are OK
Line 59: Line 66:
 
* BBa_K1707012
 
* BBa_K1707012
  
−
SpeI + Pst
+
SpeI + Pst:
−
=== New culture===
+
* BBa_R0051 x2
−
'' by Pauline''
+
* BBa_K1707013
 +
* BBa_K1707019
 +
* BBa_K1707000
  
−
Biobricks:
+
XbaI + EcoRI:
−
* BBa_K1707008
+
* BBa_I13602
−
* BBa_I13600 (2014)
+
  
−
6 clones of each in 5mL LB + 20µg/mL of chloramphenicol
+
EcoRI + SpeI:
 +
* BBa_K1707004
  
−
Incubation at 37°C, 250 rpm
+
Mix:
 +
* 1 µL Enzyme 1
 +
* 1 µL Enzyme 2
 +
* 2 µL Buffer FastDigest 10x
 +
* 6 µL H2O
 +
* 10 µL Plasmide
 +
 
 +
===Purification agarose gel===
 +
''by Coralie and Audrey''
 +
 
 +
* BBa_K1707011
 +
* BBa_K1707004
 +
* BBa_E0022
 +
* BBa_E0422
 +
* BBa_K1707012
 +
* BBa_I13602
 +
 
 +
Agarose gel 1%
 +
 
 +
Migration 100 V
 +
[[File:ParisSaclay 04.08.15-Purification.jpg|400px|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_K1707011, 3. BBa_K1707004, 4. BBa_K1707004, 5. BBa_E0022, 6. BBa_E0422, 7. BBa_K1707012, 8. BBa_I13602, 9. Empty, 10. Empty</i></p></html>
 +
We can conclude that I13602 isn't digested
 +
 
 +
===Purification===
 +
''by Pauline''
 +
 
 +
* BBa_K1707011
 +
* BBa_K1707004
 +
* BBa_E0022
 +
* BBa_E0422
 +
* BBa_K1707012
 +
* BBa_R0051
 +
* BBa_K1707013
 +
* BBa_K1707019
 +
* BBa_K1707000
 +
* BBa_K1707004
 +
 
 +
With Macherey-Nagel Kit
  
 
===Soil experiment===
 
===Soil experiment===
 
''by Coralie''
 
''by Coralie''
 +
 
====Water====
 
====Water====
 
Sea Water separate in 5mL in 50mL Falcon
 
Sea Water separate in 5mL in 50mL Falcon
Line 81: Line 130:
 
Water + concentrate cells solution 10 fold dilution
 
Water + concentrate cells solution 10 fold dilution
 
Water + concentrate cells solution 100 fold dilution
 
Water + concentrate cells solution 100 fold dilution
−
We use specific antibiotic for each strain.
+
 
 +
J0: we take 100µL of each water and put it on plates with specific antibiotic:
  
 
* MacConkey + Spectinomycin
 
* MacConkey + Spectinomycin
 
* MacConkey + Tetracyclin
 
* MacConkey + Tetracyclin
 
* MacConkey + Chloramphenicol
 
* MacConkey + Chloramphenicol
−
And Just MacConkey for the Water + LB
 
  
−
====Soil====
+
And only MacConkey for the Water + LB
−
Observation of plates (31/07/2015): 
+
−
 
+
−
* Soil + H2O on new MCK:  lots of colonies are visible
+
−
* Soil + LB on new MCK: lots of colonies are visible
+
−
* LB on new MCK: nothing
+
−
* MCK alone: nothing
+
−
 
+
−
We can suppose two different hypothesis:
+
−
* The soil is contaminated
+
−
* the MCK isn't OK for our experiment: now we add lactose in the preparation. We will try with sorbitol instead of lactose.
+
−
 
+
−
We try soil + H2O and soil +LB on:
+
−
* MCK + Sorbitol: Without antibiotics, with Spectinomycine, Tetramycine or Chloramphenicol
+
−
* MCK + Lactose: Without antibiotics, with Spectinomycine, Tetramycine or Chloramphenicol
+
  
 
Incubation ON, 37°C
 
Incubation ON, 37°C
  
−
===Digestion===
+
====Soil====
−
''by Audrey and Coralie''
+
Observation of plates (03/08/2015): 
  
−
Biobricks:
+
Less contamination on plates with MCK+Sorbitol thant MCK+Lactose.
−
* BBa_K1707011 #1 to #6
+
−
* BBa_K1707021 #1 to #6
+
−
* BBa_K1707004 #1 to #6
+
−
 
+
−
Mix:
+
−
* 2 µL Plasmid
+
−
* 1 µL Buffer FastDigest 10x
+
−
* 0,5 µL XbaI
+
−
* 0,5 µL PstI
+
−
* 6 µL H2O
+
  
 +
We choose to only use MCK+Sorbitol now
  
 +
===Low Budget Challenge===
 +
''by Coralie''
  
 +
Observation of plates (03/08/2015):
 +
We can saw a little bit of colony. We can suppose that the initial culture wasn't enough in growing phase.
  
 +
We try another time, with the same protocol than the 03/08/2015 but a fresh culture of bacteria.
  
 
'''Member present:'''
 
'''Member present:'''

Latest revision as of 21:30, 18 September 2015

Tuesday 4th August

Lab Work

Plasmid extraction

by Pauline

  • BBa_K1707008 #1 to #6
  • BBa_I13600 #1 and #2

With the Macherey-Nagel Extraction kit

Digestion Verification

by Pauline and Coralie

  • BBa_K1707008 #1 to #6

Mix:

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

Incubation 2h, 37°C


Electrophoresis

by Pauline

Agarose gel 1% Migration 90V

ParisSaclay 04.08.15-Digestion vérif 008.jpg

Verification by digestion with XbaI and PstI of BBa_K1707008, from left to right: 1. DNA Ladder, 2. #1, 3. #2, 4. #3, 5. #4, 6. #5, 7. #6, 8. Empty, 9. Empty, 10. Empty, 11. Empty, 12. Empty

We can conclude that all BBa_K1707008 clones are OK

We make a glycerol stock of #1 and #2


Electrophoresis Verification

by Pauline and Coralie

Agarose gel, 1%

Migration 100V

Paris Saclay-03.08.2015 - digestion verification 2.jpg

Verification, from left to right: 1. DNA Ladder, 2. BBa_K1707021#1, 3. BBa_K1707021#2, 4. BBa_K1707021#3, 5. BBa_K1707021#4, 6. BBa_K1707021#5, 7. BBa_K1707021#6, 8. BBa_K1707011#4, 9. BBa_K1707011#5, 10. BBa_K1707011#6

Paris Saclay-03.08.15 - digestion verification 1.jpg

Verification, from left to right: 1. DNA Ladder, 2. BBa_K1707004#1, 3. BBa_K1707004#2, 4. BBa_K1707004#3, 5. BBa_K1707004#4, 6. BBa_K1707004#5, 7. BBa_K1707004#6, 8. BBa_K1707011#1, 9. BBa_K1707011#2, 10. BBa_K1707011#3, 11. Empty, 12. Empty

We can conclude that:

  • BBa_K1707021: #2, #3, #4 and #6 are OK
  • BBa_K1707011: #1 to #6 are OK
  • BBa_K1707004: #1 to #6 are OK

We make glycerol stock of them.

Digestion

by Coralie

XbaI + PstI:

  • BBa_K1707011
  • BBa_K1707004 x2
  • BBa_E0022
  • BBa_E0422
  • BBa_K1707012

SpeI + Pst:

  • BBa_R0051 x2
  • BBa_K1707013
  • BBa_K1707019
  • BBa_K1707000

XbaI + EcoRI:

  • BBa_I13602

EcoRI + SpeI:

  • BBa_K1707004

Mix:

  • 1 µL Enzyme 1
  • 1 µL Enzyme 2
  • 2 µL Buffer FastDigest 10x
  • 6 µL H2O
  • 10 µL Plasmide

Purification agarose gel

by Coralie and Audrey

  • BBa_K1707011
  • BBa_K1707004
  • BBa_E0022
  • BBa_E0422
  • BBa_K1707012
  • BBa_I13602

Agarose gel 1%

Migration 100 V

ParisSaclay 04.08.15-Purification.jpg

Verification of gel purification, from left to right: 1. DNA Ladder, 2. BBa_K1707011, 3. BBa_K1707004, 4. BBa_K1707004, 5. BBa_E0022, 6. BBa_E0422, 7. BBa_K1707012, 8. BBa_I13602, 9. Empty, 10. Empty

We can conclude that I13602 isn't digested

Purification

by Pauline

  • BBa_K1707011
  • BBa_K1707004
  • BBa_E0022
  • BBa_E0422
  • BBa_K1707012
  • BBa_R0051
  • BBa_K1707013
  • BBa_K1707019
  • BBa_K1707000
  • BBa_K1707004

With Macherey-Nagel Kit

Soil experiment

by Coralie

Water

Sea Water separate in 5mL in 50mL Falcon Fresh water separate in 5mL in 50mL Falcon Contamination with 1mL Water + LB Water + concentrate cells solution Water + concentrate cells solution 10 fold dilution Water + concentrate cells solution 100 fold dilution

J0: we take 100µL of each water and put it on plates with specific antibiotic:

  • MacConkey + Spectinomycin
  • MacConkey + Tetracyclin
  • MacConkey + Chloramphenicol

And only MacConkey for the Water + LB

Incubation ON, 37°C

Soil

Observation of plates (03/08/2015):

Less contamination on plates with MCK+Sorbitol thant MCK+Lactose.

We choose to only use MCK+Sorbitol now

Low Budget Challenge

by Coralie

Observation of plates (03/08/2015): We can saw a little bit of colony. We can suppose that the initial culture wasn't enough in growing phase.

We try another time, with the same protocol than the 03/08/2015 but a fresh culture of bacteria.

Member present:

  • Instructors: Claire
  • Students: Coralie, Audrey and Pauline

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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.