User:DavidC/25 September 2009
From 2009.igem.org
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Friday the 25th
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).