Team:Paris Saclay/Measurement

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Measurement

1st July

Rehydratation : I13504 J23117 J23106 J23101

2nd July

Transformation : I13504 J23117 J23106 J23101

3rd July

Liquide culture : I13504 J23117 J23106 J23101

8th July

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

9th July

Transformation:

   BBa_J23101 + BBa_I13504
   BBa_J23106 + BBa_I13504
   BBa_J23117 + BBa_I13504

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

15th July

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.

16th July

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

17th July

New culture on Plate

BBa_J23101 + BBa_I13504 BBa_J23106 + BBa_I13504 BBa_J23117 + BBa_I13504

23rd July

Liquid culture from the 3 stocks

24th July

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

28th July

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

.=29th July=

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

30th July

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.