In-Fusion® HD Cloning Kit, Restriction enzymes and X- α-gal were purchased from Takara Shuzo Co., Ltd.
The DNA purification kit, restriction endonucleases, T4 DNA ligase, pfu and LA Taq DNA polymerase with GC buffer and dNTPs were purchased from Takara.
The Quick Ligation Kit and restriction enzymes with their corresponding buffers were purchased from New England Biolabs (Ipswich, MA, USA).
PCR fragments were column purified (Qiagen Minelute kit) before restriction with BglII (Biolabs).
Another commercially available kit uses restriction enzymes to fragment DNA; probes specific to the ends of desired fragments are used to amplify the desired sequence (Olink Genomics).
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Positive clones were verified with plasmid isolation (Wizard plasmid purification kit) and EcoRI restriction cutting followed by agarose gel electrophoresis.
This is a convenient uracil-N-glcosylase-ready kit which permits restriction of any carryover amplicons with uracil glycosylase, should this be required.
DNA cloning kit, RNase A, restriction enzymes (BamHI, NotI, and EcoRI), T4-ligase, and Proteinase K were purchased from Fermentas (Thermo Fisher Scientific Inc., Waltham, USA).
The PCR products were isolated by agarose electrophoresis and extracted from agarose gel using the DNA gel extraction kit, digested with restriction enzymes NdeI and XhoI respectively, and re-cloned into the vector pET-28a digested with NdeI and XhoI, respectively.
