Team:Evry/Interlab Study/09-10-2014

Construction n°1: PSB1C3 with I20260

Construction n°2: PSB1C3 with J23101-E1010

Construction n°3: PSB1C3 with K823012-E1010

Other constructions of the Anderson library of constitutive promoters

Miniprep disgestion of PSB1C3+E0240 plasmid.

• 1 µl EcoRI
• 1 µl XbaI
• 2 µl buffer 2.1
• 0.2 µl BSA
• 5 µl template (miniprep)
• 10.8 µl miliQ watter

Miniprep disgestion of BBa_J61002+J23101, BBa_J61002+J23118, BBa_J61002+J23105, BBa_J61002+J23106 and BBa_J61002+J23107 plasmids.

• 1 µl EcoRI
• 1 µl SpeI
• 2 µl buffer 2.1
• 0.2 µl BSA
• 5 µl template (miniprep)
• 10.8 µl miliQ watter

Incubation 45 minutes at 37°C. Denaturation step 20 minutes at 80 °C.

Ligation to obtain constructions PSB1C3+E0240+J23101, PSB1C3+E0240+J23118, PSB1C3+E0240+J23105, PSB1C3+E0240+J23106 and PSB1C3+E0240+J23107.

• 1 µl T4 DNA ligase
• 6 µl Insert (digested BBa_J61002+J23101, BBa_J61002+J23118, BBa_J61002+J23105, BBa_J61002+J23106 and BBa_J61002+J23107)
• 2 µl vector (digested PSB1C3+E0240)
• 2 µl buffer
• 9 µl miliQ watter

Incubation 45 minutes at room temperature.
Transformation in E. coli DH5 alpha with the protocol of the 15th of August. Bacteria were plated on Chloramphenicol agar plates and incubated at 37°C overnight.

Verification of amplification
Sep 10