Contents 1 Notebook : July 16 1.1 Lab work 1.1.1 A - Aerobic/Anaerobic regulation system 1.1.1.1 Objective : obtaining BBa_K1155003, BBa_K1155007 1.1.1.2 1 - Extraction of BBa_B0015, BBa_B0017, BBa_I732019 from DH5α 1.1.1.3 2 - Digestion of BBa_B0015, BBa_B0017, BBa_I732019 by EcoRI/PstI 1.1.1.4 Objective : obtaining biobricks in PSB3K3 1.1.1.5 1 - Digestion of pSB3K3 by EcoRI/PstI 1.1.1.6 2 - Electrophoresis of the digestion of PSB3K3 by EcoRI/PstI 1.1.1.7 3 - Gel purification of electrophoresis of the digestion of pSB3K3 by EcoRI/PstI 1.1.2 B - PCB sensor system 1.1.2.1 Objective : obtaining BBa_K1155001, BBa_K1155002, BphR2 in pSB1C3 1.1.2.2 1 - Electrophoresis of the digestion of BBa_K1155001, BBa_K1155002, BphR2 in pSB1C3

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.p

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

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_K1155002 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 : 921bp

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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