Assembly Methods




Assembly PCR

The assembly PCR has been carried out in a volume of 50 ul containing 200 uM of each dNTP, 0.1 uM of each oligonucleotide, 0.5 U of Taq DNA polymerase (Promega) in a buffer containing 2.5 mM MgCl2. Assembly PCR was conducted as follows: denaturation at 95°C for 2 min followed by 30 cycles at 95°C for 30 s, 52°C for 30 s and 72°C for 1 min, and terminated by an incubation at 72°C for 5 min.


LCR

The LCR has been carried out in a final volume of 25 ul containing 0.1 uM of each oligonucleotides phosphorylated using the T4 Polynucleotide Kinase (NEB), and 10 U of Taq DNA Ligase (NEB). LCR was conducted as follows: 94°C for 2 min; 40 cycles at 94°C for 30 s, 51°C for 4 min.


Second PCR

The full length product from LCR or assembly PCR reactions has been PCR amplified using primers:
yb5-EcoRI-5'    5'-GCGCGCGAATTCCATGCCTAAAGTTTACAGTTACCAAGAAGTTGC-3'
yb5- SacI-3'    5'-GCGCGCGAGCTCTTATTCGTTCAACAAATAATAAGCAACACCTAG- 3'
in a 50 ul reaction containing 3 ul of assembly PCR mix or 5 ul of LCR mix, 200 uM of each dNTP, 1 uM of each primer, 0.5 U of Taq DNA polymerase (Promega) and a buffer containing 2.5 mM of MgCl2. PCR was conducted as follows: denaturation at 95°C for 2 min followed by 30 cycles at 95°C for 30 s, 55°C for 30 s and 72°C for 1 min, and terminated by an incubation at 72°C for 5 min.