Difference between revisions of "Team:Paris Saclay/Notebook/July/28"
(→Tuesday 28th July) |
|||
Line 125: | Line 125: | ||
''by Johan'' | ''by Johan'' | ||
− | + | ||
+ | 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 | ||
We create new culture of BBa_J23101/BBa_J23106/BBa_J23117 + BBa_I13504. | We create new culture of BBa_J23101/BBa_J23106/BBa_J23117 + BBa_I13504. |
Revision as of 15:00, 7 August 2015
Contents
Tuesday 28th July
Lab Work
Plasmid extraction
by Coralie
Biobricks:
- BBa_K1707005 #1 and #2
- BBa_K1707006 #1 and #2
- BBa_K1707007 #1 and #2
With Macherey-Nigel Nucleospin kit
Digestion
by Coralie
- BBa_B0030 and BBa_R0051
- 2µL Buffer FastDigest 10x
- 1µL SpeI
- 1µL PstI
- 10µL Plasmid
- 6µL H2O
- BBa_K1707007, BBa_K1707006, BBa_K1399019 and BBa_K1399023
- 2µL Buffer FastDigest 10x
- 1µL XbaI
- 1µL PstI
- 10µL Plasmid
- 6µL H2O
- BBa_J23117
- 1µL Buffer FastDigest 10x
- 1µL XbaI
- 1µL PstI
- 2µL Plasmid
- 5µL H2O
Incubation 1h30, 37°C
Purification gel
by Pauline, Audrey and Coralie
Biobricks:
- BBa_K1707006
- BBa_K1707007
- BBa_K1399019
- BBa_K1399023
Purification on agarose gel 1%, migration 100V
Cut with a scalpel
Purification with the PCR Clean up kit from Macherey-Nagel
Observation: BBa_K1399019 and BBa_K1399023 weren't on the right length on the gel
Soil experiment
by Audrey, Johan and Coralie
Soil
We put 100µL of each culture on MacConkey plate with specific antbiotic (Control +):
- 1696: Tetracyclin
- 1693: Spectinomycine
- 1320: Chloramphenicol
We put 30 mL of each culture in 60g of non sterile soil in a pot.
- 1696: 3 pots
- 1693: 3 pots
- 1320: 3 pots
We put 30 mL of clean LB in 60g of non sterile soil in a pot (Control -)
- LB: 3 pots
After this contamination, we take 1g of each pot to make the J0 measurement. We dilute it in 5mL of sterile H2O, and shake it during 5 min. Then, we let it to decant during 15 min. We take 50µL of the supernatant, and dilute it in 950µL of Sterile H2O (complete dilution from the soil is now at 10-2) We put 100µL of this previous dilution on plates that fits. We incubate plates over night at 37°C
Pots are placed in trays: 1 strain by tray. Trays are covered by a shrink-wrap, and we let them in room temperature
Water
We test two different water: seawater, from the Atlantic ocean, and stagnant water from Orsay's pool. For each water, we make dilutions: 10-1, 10-2, 10-3 and 10-4 We 100µL of different dilution on each type of plate:
- LB without antibiotic
- LB + Spectinomycin
- LB + tetracyclin
- LB + Chloramphenicol
- MacConkey without antibiotic
- MacConkey + Spectinomycin
- MacConkey + Tetracyclin
- MacConkey + Chloramphenicol
We incubate plates over night at 37°C
New culture
by Pauline
Biobricks:
- BBa_K1707008
- BBa_K1707010
We take 2 colony of each, and put it in 5mL of LB + 20µg/mL Chloramphenicol Incubation 37°C, 250 rpm
Purification
by Audrey
Biobricks:
- BBa_B0030
- BBa_R0051
- BBa_J23117 #1 and #2
- BBa_K1399019
- BBa_K1399023
- BBa_K1707006
- BBa_K1707007
With PCR CLean-up/Gel extraction kit from Macherey-Nigel
Quantification
by Audrey
Agarose gel 1% Migration 100V
Interlab study
by Johan
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
We create new culture of BBa_J23101/BBa_J23106/BBa_J23117 + BBa_I13504.
Member present:
- Instructors: Alice
- Students: Coralie, Audrey, Pauline, Johan and Seong-Koo