Minibest universal genomic dna extraction kit ver 5

DNA was extracted from shoots according to the instructions of MiniBEST Universal Genomic DNA Extraction Kit Ver.5.0 (TaKaRa, Japan). To examine the presence of the insertion in three barley genotypes, upstream fragments of the HvAACT1 encoding region were amplified by PCR using KOD-FX (Toyobo, Japan) from the genomic DNA. PCR primers (5’-GGTCCAACACTCTACCCTTCCTT-3’and5’-GGTGCGAGTTGCCCCTAGCTATTACAGA-3′) were used as showed by Fujii et al. [23 (link)]. The PCR products were separated by electrophoresis of 120 V running for 30 min on a 1% agarose gel using 1 × TAE buffer and stained with 4S green plus nucleic acid stain (Sangon Biotech, Shanghai, China).

Blood samples were collected from the clinical laboratory of the Seventh Affiliated Hospital of Sun Yat-sen University under an approved Institutional Review Board protocol. Genomic DNAs were purified by using the TaKaRa MiniBEST Universal Genomic DNA Extraction Kit Ver.5.0 (TaKaRa, Shiga, Japan). For each PCR reaction described below, around 1 μg total DNA was used.

C. umbratile juveniles were collected from South China Sea (Longitude 5°20.267′ N and latitude 109°48.435′ E) in September 2014 and directly frozen. Muscle tissues were used for DNA extraction according to the Genomic DNA Extraction Kit’s instructions (TaKaRa MiniBEST Universal Genomic DNA Extraction Kit Ver.5.0, Japan). The quantity (concentration) of isolated total DNA was determined by NANODROP 2000 spectrophotometer (Thermo Scientific, USA). Furthermore, quality of extracted DNA was assessed by electrophoresis on a 1% agarose gel stained with Gel Red™ (Biotium).

Trizol (Invitrogen, Carlsbad, CA, USA) was adopted to extract total RNA from MVN, and the concentration and purity of the extracted RNA were detected by a nanodrop2000 micro ultraviolet spectrophotometer (1011U, nanodrop, USA). Next, 2 μg total RNA was added with 3 U/μg RNase R (Epicenter Technologies, Madison, WI, USA) and incubated at 37 °C for 30 min. RNeasy MinElute Cleaning kits (Qiagen GmbH, Hilden, Germany) were used for purification. The expression of circ_0000811 was detected after reverse transcription to obtain cDNA and observed by agarose gel electrophoresis. Genomic DNA (gDNA) of MVN was extracted with MiniBEST Universal Genomic DNA Extraction Kit Ver.5.0 (Takara, Japan). Based on the designed convergent polymer primers and divergent primers, circ_0000811 was detected in cDNA and gDNA and observed by agarose gel electrophoresis.

Purified Bm-SP142 was incubated with virus, which was subsequently used to infect silkworms, and the level of viral DNA in virus-infected silkworms was determined by qPCR. Briefly, 200 μl of BmBDV or BmNPV was incubated with 200 μl of purified Bm-SP142 for 24 h. Meanwhile, 200 μl of the same viral titre of BmBDV or BmNPV was incubated with 200 μl of blank eluted solution for 24 h as a control. Next, 5 μl of BmBDV was orally administered to 5th instar larvae or subcutaneously injected into 5th instar larvae. Silkworms were infected with the same virus treated with blank eluted solution (control group). A total of 30 silkworms were included in each group.
After cultivation for 48 h with fresh mulberry, silkworms were dissected to collect midgut samples, and total DNA was extracted using a MiniBEST Universal Genomic DNA Extraction Kit Ver.5.0 (TaKaRa). Primer pair GP64-F and GP64-R were used to amplify the BmNPV GP64 gene from the extracted DNA by qPCR, and primer pair ns1F and ns1R were used to amplify ns1. The amplified DNA fragments were used to indicate the abundance of viral DNA in virus-infected insects.

Genomic DNA was isolated with MiniBEST Universal Genomic DNA Extraction Kit Ver.5.0 (Takara, Japan), and total RNA was isolated by using RNAiso Plus (Takara, Janpan) according to the manufacturer’s instructions. For RNase R digestion, 2 μg of total RNA was incubated for 30 min at 37 °C with or without RNase R (3 U/μg) (Epicentre Technologies, USA), and the treated RNA was subsequently purified with the RNeasy MinElute Cleanup Kit (Qiagen, Germany). The nuclear and cytoplasmic fractions were extracted using a PARIS™ Kit (Life Technologies, USA) according to the manufacturer’s protocol. cDNA was synthesized using the PrimeScript RT Reagent Kit (Takara, Japan), and RT-PCR was performed on a Quantstudio™ DX system (Applied Biosystems, Singapore) using TB Green Premix Ex Taq II (Takara, Japan). GAPDH and small nuclear U6 were used as internal controls. The sequence information of primers was listed in Additional file 1: Table S1.

Genomic DNA was isolated from whole blood using the MiniBEST Universal Genomic DNA Extraction Kit Ver.5.0 (Takara) according to the manufacturer’s instruction. The S267F polymorphism of NTCP was determined using the polymerase chain reaction–restriction fragment length polymorphism (PCR-RFLP) method as described^{19 (link)}.