Team:Paris Saclay/Notebook/July/23

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Notebook : July 23

Lab work

A - Aerobic/Anaerobic regulation system

Objective : obtaining BBa_K1155003, BBa_K1155007

1 - Electrophoresis of the digestion of BBa_I732017 by EcoRI/SpeI, BBa_K592009 by SpeI/PstI, BBa_B0015, BBa_B0017 by XbaI/PstI

Abdou, Anaïs, Xavier, Zhou

Psgellast2307.jpg
  • Well 1 to 3: 30µL of BBa_I732017 digested by EcoRI/SpeI+6µL of 6X loading dye
  • Well 4 : 6µL of DNA 6X loading dye
  • Gel : 0.8%
Psgel22307.jpg
  • Well 1 : 5µL of BBa_B0015 digested by XbaI/PstI+1µL of 6X loading dye
  • Well 2 : 5µL of BBa_B0017 digested by XbaI/PstI+1µL of 6X loading dye
  • Well 3 : 5µL of BBa_K592009 digested SpeI/PstI+1µL of 6X loading dye
  • Well 4 : 6µL of DNA 6X loading dye
  • Gel : 0.8%

Expected sizes :

  • RBS-LacZ : 3093bp
  • Term : 150bp
  • pSB3A2 : 2079bp

We obtain a RBS-lacZ and Term fragments at the right size. The digestions were good and we will purify them.

2 -Gel purification of digestion products : RBS-LacZ

Xavier

Protocol : [http://www.mn-net.com/tabid/1452/default.aspx Gel purification ]

3 - Electrophoresis to check the gel purification of digestion products : RBS-LacZ

Abdou

Psgel32307.jpg
  • Well 1 : 6µL of DNA 6X loading dye
  • Well 2: 5µL of RBS-LacZ+1µL of 6X loading dye
  • Gel : 0.8%

Expected sizes :

  • RBS-LacZ : 3093bp

We can't see anything. The purification wasn't good. We will do the digestion again.

4 - Digestion of BBa_I732017 by EcoRI/PstI

Anaïs

Used quantities :

  • BBa_I732017 : 10µL
  • Buffer FD : 3µL
  • EcoRI FD : 1.5µL
  • PstI FD : 1.5µL
  • H2O : 14µL

5 - Electrophoresis to check the gel digestion of BBa_I732017 by EcoRI/SpeI

Zhou

Psgel42307.jpg
  • Well 1 : 6µL of DNA 6X loading dye
  • Well 2 to 4 : 5µL of BBa_I732017 digested by EcoRI/SpeI+1µL of 6X loading dye
  • Gel : 0.8%

Expected sizes :

  • RBS-LacZ : 3093bp

We can't see anything. The digestion wasn't good, we will do it the again.

6 - Digestion of BBa_K592009 by SpeI/PstI

Anaïs

Used quantities :

  • Buffer FD : 3µL
  • DNA : 15µL
  • SpeI FD : 2µL
  • PstI : 2µL
  • H2O : 8µL

7 - Electrophoresis to check the digestion of BBa_K592009 by SpeI/PstI

Zhou

Psgel12307.jpg
  • Well 1 : 6µL of DNA 6X loading dye
  • Well 2 to 4 : 5µL of BBa_K592009 digested by SpeI/PstI+1µL of 6X loading dye
  • Gel : 0.8%

We can't see anything. The purification wasn't good. We will do cultures again.

8 - Liquid culture of BBa_I732017 and BBa_K592009 in DH5α

Abdou, Xavier

We let our preparations at 37°C, 150rpm with 5mL of LB and 5mL of chloramphenicol antibiotic.

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