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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 OK

We 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 digested

Purification

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

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