Difference between revisions of "Team:Paris Saclay/Notebook/August/7"

(Created page with "=Friday 7th August= ==Lab Work== ===Soil experiment=== ''by Coralie'' ====Water==== Observation of plates (05/08/2015) Almost all plates are yellow. We choose to stop the exper...")
 
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==Lab Work==
 
==Lab Work==
  
−
===Soil experiment===
+
===Transformation===
 
''by Coralie''
 
''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''
 
  
 
Biobricks:
 
Biobricks:
−
* BBa_K1707012
+
* BBa_K1707032
−
* BBa_K1707004
+
* BBa_K1707033
−
* BBa_K1707011
+
* BBa_K1707026
−
* BBa_R0051
+
−
* BBa_K1707008
+
−
* BBa_I13602
+
−
* BBa_I13600
+
−
 
+
−
And PIJ773 digested by NotI
+
−
 
+
−
Agarose gel 1%
+
  
−
Migration 90 V
+
As usual
  
−
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===
+
===Plasmid extraction===
 
''by Pauline''
 
''by Pauline''
  
 
Biobricks:
 
Biobricks:
−
* BBa_K1707011 #1
+
* BBa_R0040 #1 and #2
−
* BBa_K1707004 #1
+
* BBa_K1707020 #1 and #2
−
* BBa_K1707012 #1
+
* BBa_K1707027 #1 and #2
−
* BBa_R0051
+
* BBa_K1707028 #1 and #2
−
* BBa_K1707008 #1
+
* Plasmid test #1 and #2
−
* BBa_I136000
+
−
* BBa_I13602
+
  
−
With Macherey-Nagel Kit
+
With Macherey-Nagel Extraction kit
  
  
−
===Quantification===
+
===Digestion verification===
−
'' by Pauline and Audrey''
+
''by Pauline''
  
 
Biobricks:
 
Biobricks:
−
* BBa_K1707011 #1
+
* BBa_K1707020 #1 and #2
−
* BBa_K1707004 #1
+
* BBa_K1707027 #1 and #2
−
* BBa_K1707012 #1
+
* BBa_K1707028#1 and #2
−
* BBa_R0051
+
* Plasmid test #1 and #2
−
* BBa_K1707008 #1
+
−
* BBa_I136000
+
−
* BBa_I13602
+
  
−
PIJ773 digested by NotI
 
  
−
Agarose gel, 1%
+
Mix:
 +
* 2 µL plasmid
 +
* 0,5 µL XbaI
 +
* 0,5 µL PstI
 +
* 1µL Buffer FastDigest 10x
 +
* 6 µL H2O
  
−
Migration 100V
+
Incubation 1h30, 37°C
  
  
−
===Ligation===
+
===SpeI Test===
−
''by Audrey''
+
''by Pauline''
−
 
+
−
Biobricks:
+
−
BBa_K1707026 : BBa_E0422 + BBa_J23101
+
  
 
Mix:
 
Mix:
−
* 11 µL BBa_E0422
+
* 2 µL BBa_K1707028 #1
−
* 8 µL BBa_J23101
+
* 0,5 µL SpeI
−
* 1,5 µL Buffer 10x
+
* 1µL Buffer N
−
* 2 µL Ligase
+
* 6,5 µL H2O
−
* 2,5 µL H2O
+
  
 +
Incubation 2h30, 37°C
  
  
−
BBa_K1707032 : BBa_I13600 + BBa_K1707008
+
===Electrophoresis===
 +
''by Pauline''
  
−
Mix:
+
Agarose gel 1%, 100V
−
* 2 µL BBa_K1707008
+
−
* 5 µL BBa_I13600
+
−
* 1 µL Buffer 10x
+
−
* 2 µL Ligase
+
  
 +
We can conlude that:
 +
* SpeI is OK
 +
* BBa_K1707020 #1 isn't OK
 +
* BBa_K1707020 #2 is OK
 +
* BBa_K1707027 #1 and #2 are OK
 +
* BBa_K1707028 #1 and #2: we have to check them another time
 +
* Plasmid test #1 and #2 are OK but not totally digested
  
 +
===New culture===
 +
''by Pauline''
  
 +
Biobrick: BBa_R0051 #1
 +
with 5mL LB and 5 µL Ampicillin
  
−
BBa_K1707033 : BBa_J23101 + BBa_K1707006
+
 
 +
===Digestion Verification ===
 +
''by Pauline''
  
 
Mix:
 
Mix:
−
* 6,5 µL BBa_K1707006
+
* 2 µL Plasmid
−
* 1,5 µL BBa_J23101
+
* 0,5 µL XbaI
−
* 1,5 µL Tampon 10x
+
* 0,5 µL PstI
−
* 2 µL Ligase
+
* 6 µL H2O
−
* 3,5 µL H2O
+
* 1 µL Buffer FastDigest 10x
  
  
−
Incubation ON, 4°C
+
===Low budget challenge===
−
 
+
''by Coralie''
−
===New culture===
+
We can see a lot of small colony. We put it ON 37°C.
−
''by Pauline''
+
−
 
+
−
Biobricks:
+
−
* BBa_K1707020
+
−
* Plamsid test
+
−
* BBa_K1707027
+
−
* BBa_R0040
+
−
* BBa_K1707028
+
−
 
+
−
2clones in 5mL LB + antibiotics
+
  
  
 
'''Member present:'''
 
'''Member present:'''
 
* Instructors: Claire
 
* Instructors: Claire
−
* Students: Coralie, Audrey and Pauline
+
* Students: Coralie and Pauline
 
{{Team:Paris_Saclay/notebook_footer}}
 
{{Team:Paris_Saclay/notebook_footer}}

Revision as of 13:05, 10 August 2015

Friday 7th August

Lab Work

Transformation

by Coralie

Biobricks:

  • BBa_K1707032
  • BBa_K1707033
  • BBa_K1707026

As usual


Plasmid extraction

by Pauline

Biobricks:

  • BBa_R0040 #1 and #2
  • BBa_K1707020 #1 and #2
  • BBa_K1707027 #1 and #2
  • BBa_K1707028 #1 and #2
  • Plasmid test #1 and #2

With Macherey-Nagel Extraction kit


Digestion verification

by Pauline

Biobricks:

  • BBa_K1707020 #1 and #2
  • BBa_K1707027 #1 and #2
  • BBa_K1707028#1 and #2
  • Plasmid test #1 and #2


Mix:

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

Incubation 1h30, 37°C


SpeI Test

by Pauline

Mix:

  • 2 µL BBa_K1707028 #1
  • 0,5 µL SpeI
  • 1µL Buffer N
  • 6,5 µL H2O

Incubation 2h30, 37°C


Electrophoresis

by Pauline

Agarose gel 1%, 100V

We can conlude that:

  • SpeI is OK
  • BBa_K1707020 #1 isn't OK
  • BBa_K1707020 #2 is OK
  • BBa_K1707027 #1 and #2 are OK
  • BBa_K1707028 #1 and #2: we have to check them another time
  • Plasmid test #1 and #2 are OK but not totally digested

New culture

by Pauline

Biobrick: BBa_R0051 #1 with 5mL LB and 5 µL Ampicillin


Digestion Verification

by Pauline

Mix:

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


Low budget challenge

by Coralie We can see a lot of small colony. We put it ON 37°C.


Member present:

  • Instructors: Claire
  • Students: Coralie and Pauline

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t@iGEMParisSaclay
aUniversité Paris Sud
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*This wiki was conceived during long nights of work and a great coffee experience in the beautiful Orsay, France.