Difference between revisions of "Team:Paris Saclay/Measurement"

Measurement

=07/01:= Rehydratation : I13504 J23117 J23106 J23101 =07/02= 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 [[File:ParisSaclay_16.07.15_-_digestion_vérif.jpg|thumb|none]] =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.