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Thursday 6th August

Lab Work

Soil experiment

by Coralie

Water

Observation of plates (05/08/2015) Almost all plates are yellow. We choose to stop the experiment.

We will define a new protocol:

• Count of bacteria before contamination
• Evaluation of the count of bacteria we should see at Day0
• Measurement of the pH before each sampling (We think that maybe the water pH change with adding the LB)

Low Budget Challenge

by Coralie

We can't say a lot of things... But we can see some colony on the agar with the baker's yeast in the agar. We try another time with different agar recipes:

• Agar 1:
  • 0,27g Stock Cube
  • 0,600g Agar Agar
  • 0,135g Baker's yeast
  • 0,225g sugar
  • 45mL mineral water

• Agar 2:
  • 0,27g Stock Cube
  • 0,600g Agar Agar
  • 0,135g Baker's yeast
  • 45mL mineral water

We make striations with BBa_J23101+BBa_I13504 strain with a curved pin. We do the same on an usual LB plate without antibiotics (Control +)

Incubation over night, 37°C

Purification agarose gel

by Pauline and Audrey

• BBa_K1707012
• BBa_K1707004
• BBa_K1707011
• BBa_R0051
• BBa_K1707008
• BBa_I13602
• BBa_I13600

And PIJ773 digested by NotI

Agarose gel 1%

Migration 90 V

Verification of gel purification, from left to right: 1. BBa_PIJ773 2. DNA Ladder, 3. BBa_K1707004, 4. BBa_K1707008, 5. BBa_K1707011, 6. BBa_K1707012, 7. BBa_R0051, 8. BBa_I13600, 9. BBa_I13602, 10. Empty

We made a mistake: BBa_K1707008, BBa_I13602 and Bba_R0051 shouldn't be purified on agarose gel. We can observe a big smear for PIJ773. We can also observe that BBa_I13602 has three bands. Rest is alright.

Purification

by Pauline

• BBa_K1707011 #1
• BBa_K1707004 #1
• BBa_K1707012 #1
• BBa_R0051
• BBa_K1707008 #1
• BBa_I136000
• BBa_I13602

With Macherey-Nagel Kit

Quantification

by Pauline and Audrey

• BBa_K1707011 #1
• BBa_K1707004 #1
• BBa_K1707012 #1
• BBa_R0051
• BBa_K1707008 #1
• BBa_I136000
• BBa_I13602

PIJ773 digested by NotI

Agarose gel, 1%

Migration 100V

Wells 2-8: Quantification, Wells 9-10: Verification; from left to right: 1. DNA Ladder, 2. BBa_K1707012#1, 3. BBa_I13602, 4. BBa_I13600, 5. BBa_K1707011#1, 6. BBa_K1707004#1, 7. BBa_K1707008#1, 8. BBa_R0051, 9. BBa_PIJ773, 10. PIJ773 plasmid

Ligation

by Audrey

BBa_K1707026:

• BBa_E0422 + BBa_J23101

Mix:

• 11 µL BBa_E0422
• 8 µL BBa_J23101
• 1,5 µL Buffer 10x
• 2 µL Ligase
• 2,5 µL H2O

BBa_K1707032:

• BBa_I13600 + BBa_K1707008

Mix:

• 2 µL BBa_K1707008
• 5 µL BBa_I13600
• 1 µL Buffer 10x
• 2 µL Ligase

BBa_K1707033:

• BBa_J23101 + BBa_K1707006

Mix:

• 6,5 µL BBa_K1707006
• 1,5 µL BBa_J23101
• 1,5 µL Tampon 10x
• 2 µL Ligase
• 3,5 µL H2O

Incubation ON, 4°C

New culture

by Pauline

• BBa_K1707020
• Plamsid test
• BBa_K1707027
• BBa_R0040
• BBa_K1707028

2clones in 5mL LB + antibiotics

Member present:

• Instructors: Claire
• Students: Coralie, Audrey and Pauline

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