BamHI restriction enzyme

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	Each fragment was amplified with a forward primer containing a BamHI restriction site and a reverse primer with a stop codon and a HindIII restriction site. After digested with BamHI and HindIII (Takara), the PCR products were cloned into expression vector, pET-32a(+) (Novagen).

Protocol tips
Each fragment was amplified with a forward primer containing a BamHI restriction site and a reverse primer with a stop codon and a HindIII restriction site. After digested with BamHI and HindIII (Takara), the PCR products were cloned into expression vector, pET-32a(+) (Novagen).

Publication protocol

The 3ABC gene of FMDV O/HKN/14/82 strain had been cloned into pMD18-T vector (Takara) and the plasmid pMD18T-3ABC served as a template for the following PCRs. The full-length 3ABC gene was dissected into 12 overlapping fragments by PCR kit (Takara). Fig. 1 showedthe construction map of 3ABC fragments. Each fragment
was amplified with a forward primer containing a BamHI
restriction site and a reverse primer with a stop codon
and a HindIII restriction site. After digested with BamHI
and HindIII (Takara), the PCR products were cloned into
expression vector, pET-32a(+) (Novagen). Each fragment
fused to the C-terminus of thioredoxin (Trx) was expressed in recombinant pET-32a(+) transformed E. coli
BL21(DE3) (Novagen). After treatment with 1mM IPTG
(BBI), bacteria were sonicated, centrifuged and the fusion
proteins were purified on Ni2+-IDA-Sepharose 6B affinity
columns (Sigma). The purified fusion proteins were separated by electrophoresis on 12% polyacrylamide–SDS gel
using a discontinuous Tris-glycine buffer system. These
fusion proteins, resolved on a 12% SDS–PAGE, were electrophoretically blotted onto nitrocellulose filters and examined for serological reactivities to well-characterized serum
samples. Western-blotting procedures were undertaken as
described previously (Sambrook et al., 1989). Sera used as
the primary antibody were diluted to 1:200. Horseradish
peroxidase (HRP)-conjugated rabbit anti-guinea pig or pig
antibodies (Sigma) were diluted 5000- or 6000-fold, respectively, prior to use as secondary antibodies; the HRP
substrate, 3,3
-diaminobenzidine tetrahydrochloride (DAB)
(Fluka) and H2O2, was used for color development"

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