Team:Paris Saclay/Notebook/July/28
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
Verification of gel purification, from left to right: 1. Empty, 2. Empty, 3. Empty, 4. DNA Ladder, 5. BBa_K1707006, 6. BBa_K1707007, 7. BBa_K1399019, 8. BBa_K1399023, 9. Empty, 10. Empty, 11. Empty, 12. Empty
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 gelSoil 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
Quantification, from left to right: 1. DNA Ladder, 2. BBa_B0030, 3. BBa_R0051, 4. BBa_J23117#1, 5. BBa_J23117#2, 6. BBa_K1399019, 7. BBa_K1399023, 8. BBa_K1707006, 9. BBa_K1707007, 10. BBa_K1707003#4
Interlab study
by Johan
https://2015.igem.org/Team:Paris_Saclay/Measurement#28th_July
Member present:
- Instructors: Alice
- Students: Coralie, Audrey, Pauline, Johan and Seong-Koo
eigem.parissaclay AT gmail DOT com
t@iGEMParisSaclay
aUniversité Paris Sud
a91405 Orsay, France
Copyright © 2015 iGEM Paris Saclay. *This wiki was conceived during long nights of work and a great coffee experience in the beautiful Orsay, France.