BglI restriction enzyme

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	Subclone A and subclone G were first digested with SacII and AscI (New England BioLabs), respectively, followed by treatment with calf intestinal alkaline phosphatase (CIAP) (TaKaRa), chloroform extraction, and isopropanol precipitation, and then restricted with BglI (TaKaRa). Subclones B to F were digested with BglI.

Protocol tips
Subclone A and subclone G were first digested with SacII and AscI (New England BioLabs), respectively, followed by treatment with calf intestinal alkaline phosphatase (CIAP) (TaKaRa), chloroform extraction, and isopropanol precipitation, and then restricted with BglI (TaKaRa). Subclones B to F were digested with BglI.

Publication protocol

Subclone A and subclone G were first digested with SacII and AscI (New England BioLabs), respectively, followed by treatment with calf intestinal alkaline phosphatase (CIAP) (TaKaRa), chloroform extraction, and isopropanol precipitation, and then restricted with BglI (TaKaRa). Subclones B to F were digested with BglI. pBAC-CMV was digested with SacII and AscI. All digestion products were then separated using 1% agarose gels, excised, and purified by using a gel extraction kit (Omega). Digested fragments A to G and pBAC-CMV were ligated overnight at 4°C, transformed into DH10B competent cells, and plated on Chl+ LB culture. Ten clones were screened by restriction fragment length polymorphism (RFLP) analysis with NcoI, StuI, or HindIII. The correct clone was named pBAC-CMV-rWIV1

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