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

(Purification)
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* BBa_K1707004
 
* BBa_K1707004
  
 +
With Macherey-Nagel Kit
  
 
===Soil experiment===
 
===Soil experiment===

Revision as of 09:59, 10 August 2015

Tuesday 4th August

Lab Work

Plasmid extraction

by Pauline

Biobricks:

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

With the Macherey-Nagel Extraction kit

Digestion Verification

by Pauline and Coralie

Biobricks: 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

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

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

Biobricks:

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

Agarose gel 1%

Migration 100 V

We can conclude that I13602 isn't digested


Purification

by Pauline

Biobricks:

  • 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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