HindIII restriction enzyme

Restriction Enzymes HindIII

Experiment
Restriction Enzymes HindIII
Product
HindIII restriction enzyme from Takara Bio Inc
Manufacturer
Takara Bio Inc

Protocol tips

Protocol tips
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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Manufacturer protocol

Download the product protocol from Takara Bio Inc for HindIII restriction enzyme below.

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