PCR2: Second PCR round to amplification of serA from the SOE product (26-08-14) with primers 5 and 6, using the iGEM PCR protocol with Q5 polymerase. The temperature of annealing is 60°C

Result: the amplification of serA is good. Clean-up of serA (PCR2) and elution with 45µL of water.

Digestion of pSB1A3 and pSB1C3 during 1h30 at 37°C. Then, inactivation of enzymes during 20’ at 80°C

Mix:

• 10µL of plasmid
• 1µL EcoRI
• 1µL PstI
• 5µL Buffer 2.1
• 33 µL H2O

Digestion of the insert (serA) during 1h at 37°C. Then inactivation of enzymes during 20’ at 80°C

Mix:

• 20µL of serA
• 1µL EcoRI
• 1µL PstI
• 5µL Buffer 2.1
• 13 µL H2O

NEB ligation:

• 2µL of digested plasmid
• 2µL of digested serA insert
• 2µL ligase buffer
• 1µL ligase (NEB)
• 13 µL H2O

Incubation overnight at 16°C

Transformation of 90µL of DH5α super competent cells with 10µL of ligation (pSB1C3-SerA or pSB1A2-SerA). The selective medium is LB + Ampicillin (Ap) 100µg/mL or LB + Chloramphenicol (Cm) 30µg/mL

pSB1C3-SerA-1 seemed good. It was sent to sequencing. It’s OK