Difference between revisions of "Team:Paris Saclay/Notebook/July/21"

(→‎Tuesday 21st July)
Line 29: Line 29:
 
* BBa_E0240
 
* BBa_E0240
 
* BBa_J06702
 
* BBa_J06702
 +
 +
200µL in 5mL LB + Antibiotic
 +
* 1696 - Bad::Tetracyclin
 +
* 1320 - ClpP::Spectinomycine
 +
* 1693 - MG5516Z1
  
 
===Plasmid extraction===
 
===Plasmid extraction===
Line 37: Line 42:
 
* BBa_R0051
 
* BBa_R0051
 
* BBa_K098997
 
* BBa_K098997
 +
 +
 +
===Digestion===
 +
''by Coralie and Johan''
 +
In each tube:
 +
* 10 µL plasmid
 +
* 1µL of each enzyme
 +
* 1 µL FastDigest Buffer (10x)
 +
* 6 µL H2O
 +
 +
* BBa_K1707001: SpeI and EcoRI
 +
* BBa_K1707002: XbaI and PstI
 +
* BBa_K1707003 #1 and #2: XbaI and PstI
 +
* BBa_J23101: SpeI and PstI
 +
* BBa_R0051: SpeI and PstI
 +
* BBa_K098997: EcoRI and SpeI
 +
 +
Incubation 1h30, 37°C
 +
 +
 +
===Observation plates from soil experiment===
 +
''by Seong-Koo and Johan''
 +
On all MacConkey plates: we can't see anything
 +
On LB+antibiotics plates: we can't see anything
 +
On LB plates without antibiotics with 10^-5 and 10^-6 dilutions: we can't see anything
 +
On LB plates without antibiotics with 10^-2, 10^^-3 and 10^-4 dilutions: we see some colonies
 +
 +
 +
===Purification on electrophoresis gel===
 +
'' by Johan and Audrey''
 +
* BBa_K1707002
 +
* BBa_K1707003 #1 and #2
 +
* BBa_K098997
 +
 +
Agarose gel, 1%
 +
Migration: 80V
 +
 +
BBa_K1707003: the digestion doesn't work. We will try other clones from the transformation
 +
 +
We cut bands of BBa_K1707002 and BBa_K098997 with a scalpel.
 +
 +
 +
===DNA Purification===
 +
''by Audrey''
 +
* BBa_K1707002
 +
* BBa_K098997
 +
* BBa_J23101
 +
* BBa_R0051
 +
* BBa_K1707001
 +
 +
With PCR Clean-Up / Gel extraction from Macherey Nigel
 +
 +
 +
===New Culture===
 +
''by Coralie''
 +
* BBa_K1707003
 +
We put in culture 10 clones of this Biobrick in 2mL LB + 2µL Cm
 +
 +
 +
===Soil Experiment===
 +
''by Johan and Coralie''
 +
We put 5g of soil samples in plates.
 +
 +
We mesure OD600 of our cultures:
 +
* 1320 - ClpP::Cm : OD600=1,32
 +
* 1696 - Bad::tetra : OD600=1,12
 +
* 1693 MG1655Z1 : OD600=1.25
 +
 +
In each plate, we put 1mL of one of this strain:
 +
* 1320
 +
* 1696
 +
* 1693
 +
in one of this dilution: 1, 0,1 or 0,01
 +
 +
At t=0, we take 1g of contamined soil, we mixed it with 5mL of steril water, we take the supernatant, dilute it at 10^-2, and put 100µL on each MacConkey plate with the right antibiotic to allow each strain to growth.
 +
 +
We incubate plates at 37°C one night
 +
 +
Control +: We put 100µL of each strain directly on their right plate, without touching the soil
 +
Control -: We extract the soil without any contamination and put 100µL of the 10^-2 dilution on a MacConkey plate
 +
 +
  
  

Revision as of 10:08, 23 July 2015

Tuesday 21st July

Lab Work

PCR

Pauline PCR Mix:

  • Buffer (5x): 30µL
  • Forward Primer: 7,5 µL
  • Reverse Primer: 7,5µL
  • dNTP: 3µL
  • DNApol GoTaq: 0,75µL
  • MgCl2: 15µL
  • H2O: 63,75µL

2µL of DNA in each tube:

  • BBa_J23101
  • BBa_K1707000

18µL from the mix in each tube

Cycle:

  • Initiation: 95°C, 2min
  • Cycle: 95°C, 1min - 61°C, 1min - 72°C, 20sec
  • Termination: 72°C, 5min


New culture

by Pauline

  • BBa_C0051
  • BBa_E0240
  • BBa_J06702

200µL in 5mL LB + Antibiotic

  • 1696 - Bad::Tetracyclin
  • 1320 - ClpP::Spectinomycine
  • 1693 - MG5516Z1

Plasmid extraction

by Coralie and Audrey

  • BBa_K1707001 #1 and #2
  • BBa_K1707002 #1 and #2
  • BBa_K1707003 #1 and #2
  • BBa_R0051
  • BBa_K098997


Digestion

by Coralie and Johan In each tube:

  • 10 µL plasmid
  • 1µL of each enzyme
  • 1 µL FastDigest Buffer (10x)
  • 6 µL H2O
  • BBa_K1707001: SpeI and EcoRI
  • BBa_K1707002: XbaI and PstI
  • BBa_K1707003 #1 and #2: XbaI and PstI
  • BBa_J23101: SpeI and PstI
  • BBa_R0051: SpeI and PstI
  • BBa_K098997: EcoRI and SpeI

Incubation 1h30, 37°C


Observation plates from soil experiment

by Seong-Koo and Johan On all MacConkey plates: we can't see anything On LB+antibiotics plates: we can't see anything On LB plates without antibiotics with 10^-5 and 10^-6 dilutions: we can't see anything On LB plates without antibiotics with 10^-2, 10^^-3 and 10^-4 dilutions: we see some colonies


Purification on electrophoresis gel

by Johan and Audrey

  • BBa_K1707002
  • BBa_K1707003 #1 and #2
  • BBa_K098997

Agarose gel, 1% Migration: 80V

BBa_K1707003: the digestion doesn't work. We will try other clones from the transformation

We cut bands of BBa_K1707002 and BBa_K098997 with a scalpel.


DNA Purification

by Audrey

  • BBa_K1707002
  • BBa_K098997
  • BBa_J23101
  • BBa_R0051
  • BBa_K1707001

With PCR Clean-Up / Gel extraction from Macherey Nigel


New Culture

by Coralie

  • BBa_K1707003

We put in culture 10 clones of this Biobrick in 2mL LB + 2µL Cm


Soil Experiment

by Johan and Coralie We put 5g of soil samples in plates.

We mesure OD600 of our cultures:

  • 1320 - ClpP::Cm : OD600=1,32
  • 1696 - Bad::tetra : OD600=1,12
  • 1693 MG1655Z1 : OD600=1.25

In each plate, we put 1mL of one of this strain:

  • 1320
  • 1696
  • 1693

in one of this dilution: 1, 0,1 or 0,01

At t=0, we take 1g of contamined soil, we mixed it with 5mL of steril water, we take the supernatant, dilute it at 10^-2, and put 100µL on each MacConkey plate with the right antibiotic to allow each strain to growth.

We incubate plates at 37°C one night

Control +: We put 100µL of each strain directly on their right plate, without touching the soil Control -: We extract the soil without any contamination and put 100µL of the 10^-2 dilution on a MacConkey plate



Member present:

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

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t@iGEMParisSaclay
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