Materials
and Methods
Amplification of CTB
Amplification
of CTB
Based
on CTB sequence (GenBank accession no. AY101181), two primers, OL-DW1
(5’CGGGATCCAACAATGATTAAATTAAAATTTGGTGTT,
BamHI site underlined) and OL-DW2 (5’GGAATTCTTAAT
TTGCCATACTAATTGC,
EcoRI site underlined), were designed for PCR-amplification
of the entire CTB coding region with two additional engineered restriction
sites. Oligonucleotides
OL-DW1 and OL-DW3 (5’CATGCCATGGTTTGCCATACTAATTGCGGCAA, NcoI
site underlined) also were used to PCR-amplify the CTB gene excluding
the
stop codon and adding BamHI and NcoI sites. For PCR
with these two pairs of
primers, plasmid pML-CTB was used as a template. PCR was carried out
with a Techne Genius thermocycler (Duxford, Cambridge, U.K.) with the
following
parameters: 25 cycles of 94°C, 30 sec; 55°C, 30 sec; 72°C,
30 sec; finally a 10-min extension at 72°C followed with 4°C
incubation until
recovery.
Materials
and Methods
