High fidelity dna polymerase

Plasmid DNA was extracted from E. coli by a standard alkaline lysis procedure with a plasmid miniprep kit (Axygen Scientific, Union City, CA, USA). Chromosomal DNA was extracted from Bt cells as previously described (Lereclus et al., 1989 (link)). Restriction enzymes and T4 DNA ligase were used according to the manufacturer's instructions (New England Biolabs, Ipswich, MA, USA). Oligonucleotide primers were synthesized by Sangon (Shanghai, China); all primer sequences are listed in Additional file 5. PCR was performed with high-fidelity DNA polymerase (Toyobo, Osaka, Japan). Amplified fragments were purified with a PCR cleanup kit (Axygen). Digested DNA fragments were separated on 1% agarose gels and extracted using a DNA gel extraction kit (Axygen). All constructs were confirmed by sequencing (Invitrogen, Carlsbad, CA, USA).

Full-length of GGTase-Iβ coding sequence was amplified from cDNA of CAL27 cells (human cancer cell line) with a high-fidelity DNA polymerase (TOYOBO, Osaka, Japan) by using standard polymerase chain reaction (PCR) techniques. The amplified gene was cloned into a pZeroBack/blunt vector (Tiangen, Beijing, China) and re-cloned into pEGFP-C1 plasmid at KpnI and XmaI sites. The constructs were confirmed by DNA sequencing. siRNAs for HDAC1, HDAC10, and HDAC11 was custom synthesized as described before [38 (link),55 (link)]. Human GGTase-Iβ siRNA was synthesized in the following sequence: GGTase-Iβ-1 5′-GGCCUCUCAUGCUUAGUUATT-3′; GGTase-Iβ-2 5′-GGAGGAAAGUGGAAUUUGUTT-3′. siRNAs and plasmid were transfected using Lipofectamine 2000 (Invitrogen, Carlsbad, CA, USA) according to the manufacturer’s instructions.

A One Step Cloning Kit and Taq polymerase were purchased from Vazyme (Nanjing, China). High-fidelity DNA polymerase was obtained from Toyobo (Osaka, Japan). K-ACET was purchased from Megazyme (Bray, Ireland). A Bacteria DNA Kit and a Plasmid Extraction Kit were purchased from Tiangen (Beijing, China). A Gel Extraction Kit was purchased from Omega (Guangzhou, China). Chitin was purchased from Sinopharm Group (Shanghai, China). Ni-NTA Superflow was obtained from Qiagen (Duesseldorf, Germany). N-acetyl glucosamine (GlcNAc), diacetylchitobiose ((GlcNAc)₂), triacetylchitotriose ((GlcNAc)₃), tetraacetylchitotetraose ((GlcNAc)₄), acetylchitopentaose ((GlcNAc)₅), and acetylchitohexaose ((GlcNAc)₆) were purchased from Qingdao Bozhihuili Biotechnology (Qingdao, China). Other chemical reagents used in this study were of analytical grade unless specifically indicated. Colloidal Chitin and carboxymethyl Chitin were prepared according to previously reported methods [50 (link),51 (link)].