Contents 1 Notebook : August 5 1.1 Lab work 1.1.1 A - Aerobic/Anaerobic regulation system 1.1.1.1 Objective : obtaining BBa_K1155003 1.1.1.2 1 - Digestion of BBa_K1155003 by EcoRI/PstI to check the ligation between RBS-Amil CP,Term and pSB1C3 1.1.1.3 2 - Electrophoresis of the digestion of BBa_K1155003 1.1.1.4 Objective : obtaining BBa_K1155007 1.1.1.5 1 - Digestion of BBa_I732017 by EcoRI/SpeI 1.1.1.6 2 -Electrophoresis of the digestion of BBa_I732017 by EcoRI/SpeI 1.1.1.7 3 - PCR of pSB1C3 1.1.1.8 3 - Electrophoresis of PCR of pSB1C3 1.1.1.9 4 - Gel purification of electrophoresis of PCR of pSB1C3 1.1.1.10 Objective : obtaining BBa_K1155004, BBa_K1155005, BBa_K1155006 1.1.1.11 1 - PCR Colonies of BBa_K1155004, BBa_K115005, BBa_K1155006 in DH5α

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

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 obtained 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

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 obtained 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
• enzyme Phusion : 0.25µL
• H2O : 36.75µL

PCR program :

Hybridation temperature gradient :

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

3 - Electrophoresis of PCR of pSB1C3

Nadia, XiaoJing

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 obtained 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 - PCR Colonies 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 took a single colony and resuspend in 10µL H2O. For each Biobrick, we did 25 PCR Colonies.

Used quantities :

PCR preparation mix for 25 different colonies including:

• • Oligo 44 : 3.5µL
  • Oligo 45 : 3.5µL
  • Buffer Dream Taq : 70µL
  • dNTP : 28µL
  • Dream Taq : 5µL
  • H2O : 590µL

PCR reaction:

• DNA : 2µL of resuspend colony
• Mix PCR  :23µL

Total volume: 25µL

PCR Program :

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