Team:Paris Saclay/Notebook/August/5

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Revision as of 17:51, 3 October 2013

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Contents

Notebook : August 5

Lab work

A - Aerobic/Anaerobic regulation system

Objective : obtaining BBa_K1155003

1 - Digestion of BBa_K1155003 by EcoRI/PstI to check the ligation between RBS-Amil CP,Term and pSB1C3

Nadia, XiaoJing

Used quantities :

  • BBa_K1155003 : 5µL
  • EcoRI FD  : 1µL
  • PstI FD : 1µL
  • Buffer FD : 3µL
  • H2O : 20µL

We incubate the digestion at 37°C during 15 minutes.

2 - Electrophoresis of the digestion of BBa_K1155003

Damir

Psgel10508.jpg
  • Well 1 : 6µL of DNA Ladder
  • Well 2 : 30µL of BBa_K1155003 from clone 9 digested by EcoRI/PstI + 6µl of 6X loading dye
  • Well 3 : 5µL of BBa_K1155003 from clone 9 +1µl of 6X loading dye
  • Well 4 : 30µL of BBa_K1155003 from clone 11 digested by EcoRI/PstI + 6µl of 6X loading dye
  • Well 5 : 5µL of BBa_K1155003 from clone 11 +1µl of 6X loading dye
  • Well 6 : 30µL of BBa_K1155003 from clone 12 digested by EcoRI/PstI +6µl of 6X loading dye
  • Well 7 : 5µL of BBa_K1155003 from clone 12 +1µl of 6X loading dye
  • Gel : 1%

Expected sizes :

  • RBS_AmilCP-Term : 824bp
  • pSB1C3 : 2070bp

We obtain fragments at the right size. We will sequence it.

Objective : obtaining BBa_K1155007

1 - Digestion of BBa_I732017 by EcoRI/SpeI

Abdou, Damir, Nadia,

Used quantities :

  • BBa_I732017 : 41µL
  • Buffer FD : 5µL
  • EcoRI : 2µL
  • SpeI : 2µL

We incubate the digestion at 37°C for 1h30.

2 -Electrophoresis of the digestion of BBa_I732017 by EcoRI/SpeI

Damir, Nadia

Psgel20508.jpg
  • Well 1 : 6µL of DNA Ladder
  • Well 2 : 40µL of BBa_I732017 digested by EcoRI/Spe I+ 8µL 6X loading dye
  • Gel : 1.5%

Expected sizes :

  • RBS-LacZ : 3093 bp
  • pSB1A2 : 2079 bp

We obtain fragments at the right size. We will make an electro-elution to extract RBS-LacZ.

3 - PCR of PSB1C3

Nadia, XiaoJing

Used quantities :

  • pSB1C3 : 2µL
  • Buffer phusion : 10µL
  • Oligo 64 : 2.5µL
  • Oligo 65 : 2.5µL
  • dNTP : 1µL
  • Phusion : 0.25µL
  • H2O : 36.75µL

PCR program :

Hybridation temperature gradient :

A - 65 B - 64.7 C - 64.1 D - 63.1 E - 62 F - 61.2 G - 60.5 H - 60

PsPCRC30508.jpg

3 - Electrophoresis of PCR of pSB1C3

Nadia, XiaoJing

Psgel30508.jpg
  • Well 1 : 6µL of DNA Ladder
  • Well 2 to 9 : 2µL of pSB1C3+3µL of H2O+1µL of 6X loading dye
  • Gel : 1%

Expected sizes :

  • pSB1C3 : 2070bp

We obtain fragments at the right size. We will purify it.

4 - Gel purification of electrophoresis of PCR of pSB1C3

Nadia, XiaoJing

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

Objective : obtaining BBa_K1155004, BBa_K1155005, BBa_K1155006

1 - Colony PCR of BBa_K1155004, BBa_K115005, BBa_K1155006 in DH5α

Damir, Nadia, XiaoJing

Transformation of 07/31/13 works. We will do a PCR Colony.

We mix our DNA in 10µL of H2O.

Used quantities :

  • DNA : 2µL
  • Mix  : (it was divided in 25 tubes for 25 different colonies for each promotor with 23µL of mix in each tube)
    • Oligo 44 : 3.5µL
    • Oligo 45 : 3.5µL
    • Buffer Dream Taq : 70µL
    • dNTP : 28µL
    • Dream Taq : 5µL
    • H2O : 590µL

PCR Program :

PsPCR0508.jpg


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