Team:Paris Saclay/Notebook/July/16

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We didn't obtain fragments at the right size. After reading the sequence again, we find an digestion site in the middle of our biobrick. We will do a Gibson assembly to modify this site.  
We didn't obtain fragments at the right size. After reading the sequence again, we find an digestion site in the middle of our biobrick. We will do a Gibson assembly to modify this site.  
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Revision as of 19:32, 26 September 2013

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Contents

Notebook : July 16

Lab work

A - Aerobic/Anaerobic regulation system

Objective : obtaining Bba_K1155003, Bba_K1155007

1 - Extraction of Bba_B0015, Bba_B0017, Bba_I732019 from DH5α

Zhou

Protocol : High copy plamid extraction

2 - Digestion of Bba_B0015, Bba_B0017, Bba_I732019 by EcoRI/PstI

Anaïs

  • DNA : 5µL
  • Buffer FD : 2µL
  • EcoRI FD : 1µL
  • PstI FD : 1µL
  • H2O : 21µL

We let our digestion 1h30 at 37°C.

Objective : obtaining biobricks in PSB3K3

1 - Digestion of PSB3K3 by EcoRI/PstI

Anaïs

Used quantities :

  • DNA : 5µL
  • Buffer FD : 2µL
  • EcoRI FD : 1µL
  • PstI FD : 1µL
  • H2O : 11µL

We let our digestion 1h30 at 37°C.

2 - Electrophoresis of the digestion of PSB3K3 by EcoRI/PstI

Sheng

[[]]
  • Well 1 : 5µL of PSB3K3 digested by EcoRI/PstI+1µL of 6X loading dye
  • Well 2 : 5µL of PSB3K3 digested by EcoRI/PstI+1µL of 6X loading dye
  • Well 3 : 6µL of DNA ladder
  • Well 4 : 5µL of PSB3K3 digested by EcoRI/PstI+1µL of 6X loading dye
  • Well 5 : 5µL of PSB3K3 digested by EcoRI/PstI+1µL of 6X loading dye
  • Gel : 1%

Expected sizes :

  • PSB3K3 : 2750bp

We obtain fragments at the right size.

3 - Gel purification of electrophoresis of the digestion of PSB3K3 by EcoRI/PstI

Abdou

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

B - PCB sensor system

Objective : obtaining Bba_K1155001, Bba_K1155002, BphR2 in PSB1C3

1 - Electrophoresis of the digestion of Bba_K1155001, Bba_K1155002, BphR2 in PSB1C3

Anaïs, Zhou

  • Bba_K1155001 :
[[]]
  • Well 1 to 5 : 2µL Bba_K1155001 digested by EcoRI/PstI+2µl of 6X loading dye
  • Well 6 and 7 : 2µL Bba_K1155001 digested by SacII+2µl of 6X loading dye
  • Well 8 : 6µL DNA Ladder
  • Gel : 1.2%

Expected size :

  • Bba_K1155001 digested by EcoRI/PstI : 2037bp + 333bp
  • Bba_K1155001 digested by SacII : 2370bp

In the first well, we obtain fragment at the right size. We will amplify Bba_K1155001.

  • Bba_K1155002 :
[[]]
  • Well 1 to 8 : 2µL Bba_K1155002 digested by EcoRI/PstI+2µl of 6X loading dye
  • Well 9 : 6µL DNA Ladder
  • Well 10 to 17 : 2µL Bba_K1155001 digested by SacII+2µl of 6X loading dye
  • Gel : 1.5%

Expected size :

  • Bba_K1155002 digested by EcoRI/PstI : ...
  • Bba_K1155002 digested by SacII : ...

We didn't obtain fragments at the right size. We will do the ligation again.

  • BphR2 in PSB1C3 :
[[]]
  • Well 1 to 8 : 2µL BphR2 in PSB1C3 digested by EcoRI/PstI+2µl of 6X loading dye
  • Well 9 : 6µL DNA Ladder
  • Well 10 to 17 : 2µL BphR2 in PSB1C3 digested by SacII+2µl of 6X loading dye
  • Gel : 1.5%

Expected size :

  • BphR2 digested by EcoRI/PstI : ...
  • BphR2 digested by SacII : ...

We didn't obtain fragments at the right size. After reading the sequence again, we find an digestion site in the middle of our biobrick. We will do a Gibson assembly to modify this site.


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