Team:Paris Saclay/Notebook/August/4
Contents
Tuesday 4th August
Lab Work
Plasmid extraction
by Pauline
- BBa_K1707008 #1 to #6
- BBa_I13600 #1 and #2
With the Macherey-Nagel Extraction kit
Digestion Verification
by Pauline and Coralie
- BBa_K1707008 #1 to #6
Mix:
- 2 µL Plasmid
- 0,5 µL XbaI
- 0,5 µL PstI
- 1 µL Buffer FastDigest 10x
- 6 µL H2O
Incubation 2h, 37°C
Electrophoresis
by Pauline
Agarose gel 1% Migration 90V
Verification by digestion with XbaI and PstI of BBa_K1707008, from left to right: 1. DNA Ladder, 2. #1, 3. #2, 4. #3, 5. #4, 6. #5, 7. #6, 8. Empty, 9. Empty, 10. Empty, 11. Empty, 12. Empty
We can conclude that all BBa_K1707008 clones are OKWe make a glycerol stock of #1 and #2
Electrophoresis Verification
by Pauline and Coralie
Agarose gel, 1%
Migration 100V
Verification, from left to right: 1. DNA Ladder, 2. BBa_K1707021#1, 3. BBa_K1707021#2, 4. BBa_K1707021#3, 5. BBa_K1707021#4, 6. BBa_K1707021#5, 7. BBa_K1707021#6, 8. BBa_K1707011#4, 9. BBa_K1707011#5, 10. BBa_K1707011#6
Verification, from left to right: 1. DNA Ladder, 2. BBa_K1707004#1, 3. BBa_K1707004#2, 4. BBa_K1707004#3, 5. BBa_K1707004#4, 6. BBa_K1707004#5, 7. BBa_K1707004#6, 8. BBa_K1707011#1, 9. BBa_K1707011#2, 10. BBa_K1707011#3, 11. Empty, 12. Empty
We can conclude that:- BBa_K1707021: #2, #3, #4 and #6 are OK
- BBa_K1707011: #1 to #6 are OK
- BBa_K1707004: #1 to #6 are OK
We make glycerol stock of them.
Digestion
by Coralie
XbaI + PstI:
- BBa_K1707011
- BBa_K1707004 x2
- BBa_E0022
- BBa_E0422
- BBa_K1707012
SpeI + Pst:
- BBa_R0051 x2
- BBa_K1707013
- BBa_K1707019
- BBa_K1707000
XbaI + EcoRI:
- BBa_I13602
EcoRI + SpeI:
- BBa_K1707004
Mix:
- 1 µL Enzyme 1
- 1 µL Enzyme 2
- 2 µL Buffer FastDigest 10x
- 6 µL H2O
- 10 µL Plasmide
Purification agarose gel
by Coralie and Audrey
- BBa_K1707011
- BBa_K1707004
- BBa_E0022
- BBa_E0422
- BBa_K1707012
- BBa_I13602
Agarose gel 1%
Migration 100 V
Verification of gel purification, from left to right: 1. DNA Ladder, 2. BBa_K1707011, 3. BBa_K1707004, 4. BBa_K1707004, 5. BBa_E0022, 6. BBa_E0422, 7. BBa_K1707012, 8. BBa_I13602, 9. Empty, 10. Empty
We can conclude that I13602 isn't digestedPurification
by Pauline
- BBa_K1707011
- BBa_K1707004
- BBa_E0022
- BBa_E0422
- BBa_K1707012
- BBa_R0051
- BBa_K1707013
- BBa_K1707019
- BBa_K1707000
- BBa_K1707004
With Macherey-Nagel Kit
Soil experiment
by Coralie
Water
Sea Water separate in 5mL in 50mL Falcon Fresh water separate in 5mL in 50mL Falcon Contamination with 1mL Water + LB Water + concentrate cells solution Water + concentrate cells solution 10 fold dilution Water + concentrate cells solution 100 fold dilution
J0: we take 100µL of each water and put it on plates with specific antibiotic:
- MacConkey + Spectinomycin
- MacConkey + Tetracyclin
- MacConkey + Chloramphenicol
And only MacConkey for the Water + LB
Incubation ON, 37°C
Soil
Observation of plates (03/08/2015):
Less contamination on plates with MCK+Sorbitol thant MCK+Lactose.
We choose to only use MCK+Sorbitol now
Low Budget Challenge
by Coralie
Observation of plates (03/08/2015): We can saw a little bit of colony. We can suppose that the initial culture wasn't enough in growing phase.
We try another time, with the same protocol than the 03/08/2015 but a fresh culture of bacteria.
Member present:
- Instructors: Claire
- Students: Coralie, Audrey and Pauline
eigem.parissaclay AT gmail DOT com
t@iGEMParisSaclay
aUniversité Paris Sud
a91405 Orsay, France
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