Difference between revisions of "Team:Paris Saclay/Notebook/July/29"

(Created page with "=Wednesday 29th July= ==Lab Work== ===Plasmid extraction=== ''by Seong-Koo'' Biobricks: * BBa_K1707008 #1 and #2 * BBa_K1707010 #1 and #2 * BBa_R0051 With the Macherey-Nagel Ex...")
 
(→‎Wednesday 29th July)
 
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{{Team:Paris_Saclay/notebook_header}}
 
=Wednesday 29th July=
 
=Wednesday 29th July=
 
==Lab Work==
 
==Lab Work==
Line 46: Line 47:
  
 
Purification on agarose gel 1%, migration 100V
 
Purification on agarose gel 1%, migration 100V
 +
[[File:ParisSaclay 29.07.15-vérif purif.jpg|300px|center]]
 +
<html><i><p>Verification of gel purification, from left to right: 1. Empty, 2. Empty, 3. Empty, 4. <a href="https://2015.igem.org/File:Paris_Saclay-Ladder.jpg" target="_blank">DNA Ladder</a>, 5. BBa_K1707010#1, 6. Empty, 7. Empty, 8. Empty, 9. Empty, 10. Empty</p></i></html>
 
Cut with a scalpel
 
Cut with a scalpel
  
Line 82: Line 85:
 
* BBa_R0051
 
* BBa_R0051
 
Agarose gel, 1%, migration 110V
 
Agarose gel, 1%, migration 110V
 +
[[File:ParisSaclay 29.07.15-quantif+vérif.jpg|300px|center]]
 +
<html><i><p>Wells 1-5: Quantification, Wells 7-8: Verification by digestion with NotI; from left to right: 1. BBa_K1707003#5, 2. BBa_K1707008#1, 3. BBa_K1707010#1, 4. BBa_R0051, 5. R0051 plasmid, 6. <a href="https://2015.igem.org/File:Paris_Saclay-Ladder.jpg" target="_blank">DNA Ladder</a>, 7. BBa_K1707005#1, 8. BBa_K1707005#2, 9. Empty, 10. Empty, 11. Empty, 12. Empty</p></i></html>
  
 
We can conclude that K1707005 isn't digested
 
We can conclude that K1707005 isn't digested
Line 91: Line 96:
 
* BBa_R0051: 8ng/µL
 
* BBa_R0051: 8ng/µL
  
 +
===Ligation===
 +
''by Audrey''
  
 +
* BBa_K1707012: BBa_B0030 + BBa_K1707007
 +
** 6 µL BBa_B0030
 +
** 13 µL BBa_K1707007
 +
** 2,5µL Buffer Ligase 10x
 +
** 1 µL Ligase
 +
** 2,5 µL H2O
  
−
===Soil experiment===
+
* BBa_K1707019: BBa_K1707000 + BBa_K1707006
−
''by Audrey, Johan and Coralie''
+
** 2,5µL BBa_K1707000
 +
** 13 µL BBa_K1707006
 +
** 2 µL Buffer Ligase 10x
 +
** 1 µL Ligase
 +
** 1,5 µL H2O
  
−
==Soil==
+
* BBa_K1707013: BBa_K1707000 + BBa_K1707007
−
We put 100µL of each culture on MacConkey plate with specific antbiotic (Control +):
+
** 1 µL BBa_K1707000
−
* 1696: Tetracyclin
+
** 6 µL BBa_K1707007
−
* 1693: Spectinomycine
+
** 1 µL Buffer Ligase 10x
−
* 1320: Chloramphenicol
+
** 1 µL Ligase
 +
** 1 µL H2O
  
−
We put 30 mL of each culture in 60g of non sterile soil in a pot.
+
* BBa_K1707009: BBa_S03518 + BBa_R0051
−
* 1696: 3 pots
+
** 5,5µL BBa_S03518
−
* 1693: 3 pots
+
** 6,5 µL BBa_R0051
−
* 1320: 3 pots
+
** 1,5µL Buffer Ligase 10x
 +
** 1 µL Ligase
 +
** 0,5 µL H2O
  
−
We put 30 mL of clean LB in 60g of non sterile soil in a pot (Control -)
+
* BBa_K1707004: BBa_K1707003 + BBa_R0051
−
* LB: 3 pots
+
** 10 µL BBa_K1707003
 +
** 6,5 µL BBa_K1707007
 +
** 2 µL Buffer Ligase 10x
 +
** 1 µL Ligase
 +
** 0,5 µL H2O
  
−
After this contamination, we take 1g of each pot to make the J0 measurement. We dilute it in 5mL of sterile H2O, and shake it during 5 min. Then, we let it to decant during 15 min.
+
Incubation over night, 16°C
−
We take 50µL of the supernatant, and dilute it  in 950µL of Sterile H2O (complete dilution from the soil is now at 10-2)
+
−
We put 100µL of this previous dilution on plates that fits.
+
−
We incubate plates over night at 37°C
+
  
−
Pots are placed in trays: 1 strain by tray. Trays are covered by a shrink-wrap, and we let them in room temperature
+
===Transformation===
 +
''by Coralie''
  
−
==Water==
+
Biobricks:
−
We test two different water: seawater, from the Atlantic ocean, and stagnant water from Orsay's pool.
+
* BBa_K1707012
−
For each water, we make dilutions: 10-1, 10-2, 10-3 and 10-4
+
* BBa_K1707019
−
We 100µL of different dilution on each type of plate:
+
* BBa_K1707013
−
* LB without antibiotic
+
−
* LB + Spectinomycin
+
−
* LB + tetracyclin
+
−
* LB + Chloramphenicol
+
−
* MacConkey without antibiotic
+
−
* MacConkey + Spectinomycin
+
−
* MacConkey + Tetracyclin
+
−
* MacConkey + Chloramphenicol
+
  
−
We incubate plates over night at 37°C
+
As usual
  
−
===New culture===
+
===Rehydratation and transformation===
−
''by Pauline''
+
''by Johan''
  
 
Biobricks:
 
Biobricks:
−
* BBa_K1707008
+
* BBa_E0022
−
* BBa_K1707010
+
* BBa_E0422
−
We take 2 colony of each, and put it in 5mL of LB + 20µg/mL Chloramphenicol
+
−
Incubation 37°C, 250 rpm
+
  
−
===Purification===
+
 
−
''by Audrey''
+
===New Culture===
 +
''by Pauline''
  
 
Biobricks:
 
Biobricks:
−
* BBa_B0030
+
* BBa_K1707008 #3, #4, #5 and #6
−
* BBa_R0051
+
−
* BBa_J23117 #1 and #2
+
−
* BBa_K1399019
+
−
* BBa_K1399023
+
−
* BBa_K1707006
+
−
* BBa_K1707007
+
  
−
With PCR CLean-up/Gel extraction kit from Macherey-Nigel
+
===Soil experiment===
 +
''by Johan and Coralie''
  
−
===Quantification===
+
====Soil====
−
''by Audrey''
+
Observation of J0 plates: there are a lot of colony in each. Contamination is alright. Controls (+) and (-) are Ok too.
 +
We take 1g of each pot of soil and we dilute it in 5mL sterile H2O.
 +
After shaking and decant, we put 100µL of supernatant on the right plate. Incubation ON 37°C
 +
 
 +
====Water====
  
−
Agarose gel 1%
+
We can't see anything on water plates. We try another type, like yesterday but without dilution and dilution 10-1.
−
Migration 100V
+
We 100µL of different dilution on each type of plate:
−
 
+
* LB without antibiotic
−
 
+
* LB + Spectinomycin
−
===Interlab study===
+
* LB + tetracyclin
−
''by Johan''
+
* LB + Chloramphenicol
−
 
+
* MacConkey without antibiotic
−
We try to use the TEKAN to mesure our fluorescence, but it doesn't work.
+
* MacConkey + Spectinomycin
 +
* MacConkey + Tetracyclin
 +
* MacConkey + Chloramphenicol
 +
Incubation ON 37°C
  
−
We create new culture of BBa_J23101/BBa_J23106/BBa_J23117 + BBa_I13504.
+
====Measurment====
 +
'By Johan'
  
 +
https://2015.igem.org/Team:Paris_Saclay/Measurement#29th_July
  
 
'''Member present:'''
 
'''Member present:'''

Latest revision as of 21:28, 18 September 2015

Wednesday 29th July

Lab Work

Plasmid extraction

by Seong-Koo

Biobricks:

  • BBa_K1707008 #1 and #2
  • BBa_K1707010 #1 and #2
  • BBa_R0051

With the Macherey-Nagel Extraction kit


Digestion

by Pauline

  • BBa_K1707008 #1
    • 2µL Buffer FastDigest 10x
    • 1µL SpeI
    • 1µL PstI
    • 10µL Plasmid
    • 6µL H2O
  • BBa_K1707010 #1
    • 2µL Buffer FastDigest 10x
    • 1µL XbaI
    • 1µL PstI
    • 10µL Plasmid
    • 6µL H2O
  • BBa_R0051
    • 1µL Buffer FastDigest 10x
    • 1µL SpeI
    • 1µL PstI
    • 2µL Plasmid
    • 5µL H2O

Incubation 1h30, 37°C

Purification gel

by Pauline

Biobricks:

  • BBa_K1707010 #1


Purification on agarose gel 1%, migration 100V

ParisSaclay 29.07.15-vérif purif.jpg

Verification of gel purification, from left to right: 1. Empty, 2. Empty, 3. Empty, 4. DNA Ladder, 5. BBa_K1707010#1, 6. Empty, 7. Empty, 8. Empty, 9. Empty, 10. Empty

Cut with a scalpel


Purification

by Pauline

Biobricks:

  • BBa_K1707008 #1
  • BBa_K1707010 #1
  • BBa_R0051

Purification with the PCR Clean up kit from Macherey-Nagel

Digestion for verification

by Audrey

Biobricks:

  • BBa_K1707005 #1 and #2

Mix:

  • 2µL plasmid
  • 0,5µL NotI
  • 1µL Buffer FastDigest 10x
  • 6,5µL H2O

Incubation 1h30, 37°C

Quantification

by Pauline

Biobricks:

  • BBa_K1707003 #5
  • BBa_K1707008 #1
  • BBa_K1707010 #1
  • BBa_R0051

Agarose gel, 1%, migration 110V

ParisSaclay 29.07.15-quantif+vérif.jpg

Wells 1-5: Quantification, Wells 7-8: Verification by digestion with NotI; from left to right: 1. BBa_K1707003#5, 2. BBa_K1707008#1, 3. BBa_K1707010#1, 4. BBa_R0051, 5. R0051 plasmid, 6. DNA Ladder, 7. BBa_K1707005#1, 8. BBa_K1707005#2, 9. Empty, 10. Empty, 11. Empty, 12. Empty

We can conclude that K1707005 isn't digested

We can conclude the quantification:

  • BBa_K1707003 #5: 10ng/µL
  • BBa_K1707008 #1: not OK
  • BBa_K1707010 #1: 10 ng/µL
  • BBa_R0051: 8ng/µL

Ligation

by Audrey

  • BBa_K1707012: BBa_B0030 + BBa_K1707007
    • 6 µL BBa_B0030
    • 13 µL BBa_K1707007
    • 2,5µL Buffer Ligase 10x
    • 1 µL Ligase
    • 2,5 µL H2O
  • BBa_K1707019: BBa_K1707000 + BBa_K1707006
    • 2,5µL BBa_K1707000
    • 13 µL BBa_K1707006
    • 2 µL Buffer Ligase 10x
    • 1 µL Ligase
    • 1,5 µL H2O
  • BBa_K1707013: BBa_K1707000 + BBa_K1707007
    • 1 µL BBa_K1707000
    • 6 µL BBa_K1707007
    • 1 µL Buffer Ligase 10x
    • 1 µL Ligase
    • 1 µL H2O
  • BBa_K1707009: BBa_S03518 + BBa_R0051
    • 5,5µL BBa_S03518
    • 6,5 µL BBa_R0051
    • 1,5µL Buffer Ligase 10x
    • 1 µL Ligase
    • 0,5 µL H2O
  • BBa_K1707004: BBa_K1707003 + BBa_R0051
    • 10 µL BBa_K1707003
    • 6,5 µL BBa_K1707007
    • 2 µL Buffer Ligase 10x
    • 1 µL Ligase
    • 0,5 µL H2O

Incubation over night, 16°C

Transformation

by Coralie

Biobricks:

  • BBa_K1707012
  • BBa_K1707019
  • BBa_K1707013

As usual

Rehydratation and transformation

by Johan

Biobricks:

  • BBa_E0022
  • BBa_E0422


New Culture

by Pauline

Biobricks:

  • BBa_K1707008 #3, #4, #5 and #6

Soil experiment

by Johan and Coralie

Soil

Observation of J0 plates: there are a lot of colony in each. Contamination is alright. Controls (+) and (-) are Ok too. We take 1g of each pot of soil and we dilute it in 5mL sterile H2O. After shaking and decant, we put 100µL of supernatant on the right plate. Incubation ON 37°C

Water

We can't see anything on water plates. We try another type, like yesterday but without dilution and dilution 10-1. We 100µL of different dilution on each type of plate:

  • LB without antibiotic
  • LB + Spectinomycin
  • LB + tetracyclin
  • LB + Chloramphenicol
  • MacConkey without antibiotic
  • MacConkey + Spectinomycin
  • MacConkey + Tetracyclin
  • MacConkey + Chloramphenicol

Incubation ON 37°C

Measurment

'By Johan'

https://2015.igem.org/Team:Paris_Saclay/Measurement#29th_July

Member present:

  • Instructors: Alice
  • Students: Coralie, Audrey, Pauline, Johan and Seong-Koo

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.