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==
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''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
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''by Pauline and Coralie''
 
''by Pauline and Coralie''
  
−
Biobricks: BBa_K1707008 #1 to #6
+
* BBa_K1707008 #1 to #6
  
 
Mix:
 
Mix:
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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
  
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Migration 100V
 
Migration 100V
−
 
+
[[File:Paris Saclay-03.08.2015 - digestion verification 2.jpg|400px|center]]
 +
<html>
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<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
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EcoRI + SpeI:
 
EcoRI + SpeI:
 
* BBa_K1707004
 
* BBa_K1707004
−
 
  
 
Mix:
 
Mix:
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* 6 µL H2O
 
* 6 µL H2O
 
* 10 µL Plasmide
 
* 10 µL Plasmide
−
 
  
 
===Purification agarose gel===
 
===Purification agarose gel===
 
''by Coralie and Audrey''
 
''by Coralie and Audrey''
  
−
Biobricks:
 
 
* BBa_K1707011
 
* BBa_K1707011
 
* BBa_K1707004
 
* BBa_K1707004
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Migration 100 V
 
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
 
We can conclude that I13602 isn't digested
−
 
  
 
===Purification===
 
===Purification===
 
''by Pauline''
 
''by Pauline''
  
−
Biobricks:
 
 
* BBa_K1707011
 
* BBa_K1707011
 
* BBa_K1707004
 
* BBa_K1707004
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* BBa_K1707004
 
* 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
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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
−
 
  
 
J0: we take 100µL of each water and put it on plates with specific antibiotic:
 
J0: we take 100µL of each water and put it on plates with specific antibiotic:
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We choose to only use MCK+Sorbitol now
 
We choose to only use MCK+Sorbitol now
−
 
  
 
===Low Budget Challenge===
 
===Low Budget Challenge===
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We try another time, with the same protocol than the 03/08/2015 but a fresh culture of bacteria.  
 
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

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.