Team:Paris Saclay/Measurement

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Measurement

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1st July

Rehydratation : I13504 J23117 J23106 J23101

2nd July

Transformation : I13504 J23117 J23106 J23101

07/03

Liquide culture : I13504 J23117 J23106 J23101

07/08

First Digestion:

   BBa_J23101
   BBa_J23106
   BBa_J23117

Mix:

   10µL of our plasmid with promotor
   1µL SpeI
   1µL PstI
   2µL buffer 10x FastDigest
   6µL H2O

Second Digestion:

   BBa_I13504

Mix:

   10µL of our plasmid with gene
   1µL XbaI
   1µL PstI
   2µL buffer 10x FastDigest

Incubation 1h30, 37°C

07/09

Transformation:

   BBa_J23101 + BBa_I13504
   BBa_J23106 + BBa_I13504
   BBa_J23117 + BBa_I13504

On LB + Chloramphenicol 20ug/mL. Incubation ON, 37°C

07/15

Liquid culture:

   BBa_J23101 + BBa_I13504
   BBa_J23106 + BBa_I13504
   BBa_J23117 + BBa_I13504

We can observe from the plate that BBa_J23101 + BBa_I13504 and BBa_J23106 + BBa_I13504 are yellow when we expose them to the UV light. But BBa_J23117 + BBa_I13504 don't seem to be yellow on the UV light.

07/16

Digestion:

   BBa_J23101 + BBa_I13504
   BBa_J23106 + BBa_I13504
   BBa_J23117 + BBa_I13504

Reaction mix:

   Plasmid: 2µL
   EcoRI: 0,5µL
   PstI: 0,5µL
   Buffer FastDigest (10x): 2µL
   H2O: 15µL

Electrophoresis:

Preparation of Agarose Gel 1%, 0,5g in 50mL of 1X TAE, 0,5µL of BET Migration 0,06A 80V

07/17

New culture on Plate

BBa_J23101 + BBa_I13504 BBa_J23106 + BBa_I13504 BBa_J23117 + BBa_I13504

07/23

Liquid culture from the 3 stocks

07/24

Cytometer

We count 500 000 events Controls:

   Cells alones
   Cells transformed by BBa_J23101, BBa_J23106 and BBa_J23117

Our measurements: Cells transformed by

   BBa_J23101 + BBa_I13504
   BBa_J23106 + BBa_I13504
   BBa_J23117 + BBa_I13504

We uses cells in growth phase and stationary phase

Between each test, we do 2 washes with bleach and 2 washes with H2O

07/28

New culture of BBa_J23101/BBa_J23106/BBa_J23117 + BBa_I13504

Tecan utilisation:

we use only LB without chloramphenicol and we suspect a contamination of our samples. We depose in inch well 300µL We analyse the OD and fluorescence's variation (the excitation and emission wave lenght were choose after a scan in the process to obtain the best results) For each sample, we depose twelve time (12x8 plate)

• LB
• Competent cells
• Cells with J23101
• Cells with J23101 + GFP
• Cells with J23106
• Cells with J23106 + GFP
• Cells with J23117
• Cells with J23117 + GFP

We let's run for 20 cycles of 1 hour

.=07/29=

Tecan utilisation:

This time, we use LB and chloramphenicol because the plate has just a protection and we suspect a contamination of our samples. We depose in inch well 300µL We analyse the OD and fluorescence's variation (the excitation and emission wave lenght were choose after a scan in the process to obtain the best results)

For each sample we use 3 different colonies, we depose 2 times each colonies (12x8 plate)

   LB
   Competent cells
   Cells with J23101
   Cells with J23101 + GFP
   Cells with J23106
   Cells with J23106 + GFP
   Cells with J23117
   Cells with J23117 + GFP

We let's run for 20 cycles of 1 hour

Flow cytometer

We analyse the sample see previously with ODmax and OD 0.4 But we doesn't use the good scale so we will reused it tomorrow

07/30

Flow cytometer

We count 500 000 events Controls:

   LB
   Cells transformed by BBa_J23101, BBa_J23106 and BBa_J23117

Our measurements: Cells transformed by

   BBa_J23101 + BBa_I13504
   BBa_J23106 + BBa_I13504
   BBa_J23117 + BBa_I13504

We uses cells in growth phase and stationary phase

Between each test, we do 2 washes with bleach and 2 washes with H2O

We use a less powerful adjustment to see tall the result than the day before.