User:DavidC/25 September 2009

Ligation
Ligation between BBa_P1003 and BBa_B0014 :

1. Plasmid (P1003) = 0,5µL 1. Insert (B0014) = 3µL

2. Plasmid (P1003) = 0,5µL 2. Insert (B0014) = 7µL

Ligation between BBa_C0012 and BBa_B0014 :

1. Plasmid (C0012) = 0,5µL 1. Insert (B0014) = 3µL

2. Plasmid (C0012) = 0,5µL 2. Insert (B0014) = 7µL

Ligation between BBa_B0030 and BBa_B0014 + BBa_J23100 :

1. Plasmid (B0030) = 1µL 1. Insert (B0014+J23100) = 4µL

2. Plasmid (B0030) = 1µL 2. Insert (B0014+J23100) = 7µL

3. Plasmid (B0030) = 0,5µL 3. Insert (B0014+J23100) = 10µL

Ligation between BBa_R0010 and BBa_B0014 :

1. Plasmid (R0010) = 0,5µL 1. Insert (B0014) = 2µL

2. Plasmid (R0010) = 0,5µL 2. Insert (B0014) = 3µL

3. Plasmid (R0010) = 0,5µL 3. Insert (B0014) = 1µL

4. Plasmid (R0010) = 1µL 4. Insert (B0014) = 5µL

Ligation between BBa_I712669 and BBa_B0014:

1. Plasmid (I712669) = 1µL 1. Plasmid (B0014) = 5µL

2. Plasmid (i712669) = 1µL 2. Plasmid (B0014) = 7µL

NEB Enzymes
For each samples add sterilized water to obtain a maximum volume equals to 8µL.

Ligation mix (NEB): 7,5µL of T4 ligase + 15µL of T4 ligase buffer. Add 1,5µL / tube. Incubation 1h at RT.

Electroporation
Electroporation cuvettes = 2mm ; inoculums of electrocompetent E.coli DH5alpha= 40µL; pulse = 2,5KVolt ; 1h of incubation.

Spread 1mL of inoculums into a petri dish with LB + ampicillin (50mg/mL) (20/0,02mL).