SYBR Premix Ex Taq (RR420A) and TRIzol Reagent (D9108B) were ordered from TaKaRa (Otsu, Shiga, Japan); a TIANamp Virus RNA/DNA Kit (DP315-R) was ordered from Tiangen (Beijing, China); a Dead Cell Apoptosis Kit with Annexin V-fluorescein (FITC) and PI (V13242), and BODIPY 493/503 (D3922) were ordered from Thermo Fisher Scientific (MA, USA); Hoechst 33342 (561908) was ordered from BD Biosciences (NJ, USA); Oil Red O (O0625) was ordered from Sigma-Aldrich (MO, USA); EGCG (HY-13653) was ordered from MedChemExpress (NJ, USA); a LabAssay nonessential fatty acid (294-63601) assay kit for FFAs was ordered from Wako Bioproducts (VA, USA); and a triglyceride assay kit (E1013) and a cholesterol assay kit (E1015) were ordered from Applygen Technologies Inc. (Beijing, China).
Tianamp virus dna rna kit
Manufactured by Tiangen Biotech
Sourced in China, Japan
The TIANamp Virus DNA/RNA Kit is a laboratory product designed for the extraction and purification of viral DNA and RNA from various sample types. It utilizes a simple and efficient silica membrane-based method to isolate high-quality nucleic acids. The kit provides a reliable and standardized solution for viral nucleic acid extraction, suitable for a wide range of downstream applications.
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Lab products found in correlation
Primescript rt reagent kit with gdna eraser, by Takara Bio (5 mentions)
Trizol reagent, by Takara Bio (3 mentions)
Tb green premix ex taq 2, by Takara Bio (3 mentions)
Bodipy 493 503, by Thermo Fisher Scientific (2 mentions)
Oil red o o0625, by Merck Group (2 mentions)
Dead cell apoptosis kit with annexin 5 fluorescein fitc and pi v13242, by Thermo Fisher Scientific (2 mentions)
Hoechst 33342 561908, by BD (2 mentions)
Sybr premix ex taq, by Takara Bio (2 mentions)
Triglyceride assay kit e1013, by Applygen (2 mentions)
Cholesterol assay kit e1015, by Applygen (2 mentions)
Tianamp genomic dna kit, by Tiangen Biotech (2 mentions)
Luna universal qpcr master mix, by New England Biolabs (2 mentions)
Wizard genomic dna purification kit, by Promega (2 mentions)
Steponeplus real time pcr system, by Thermo Fisher Scientific (2 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (2 mentions)
Primestar dna polymerase, by Takara Bio (1 mentions)
Gel doc gel imaging system, by Bio-Rad (1 mentions)
Universal dna purification kit, by Tiangen Biotech (1 mentions)
Premix ex taq probe qpcr, by Takara Bio (1 mentions)
Dnase 1, by Takara Bio (1 mentions)
101 protocols using tianamp virus dna rna kit
1
Lipid Metabolism Analysis: Detailed Reagents and Kits
2
Recombinant Virus Construction via CRISPR
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Based on the methods of Bi et al. [23 (link)], the recombinant virus with a partial UL7 deletion mutation was constructed via co-transfection of plasmids PX330-UL7-1 and PX330-UL7-2. Virus was harvested from infected 293 T cells at 48 h post-infection (p.i.), and viral genomic DNA was extracted using the TIANamp Virus RNA/DNA Kit (Tiangen, Beijing, China). The genomic region surrounding the CRISPR target site of the UL7 gene was PCR-amplified using PrimeSTAR DNA polymerase (TAKARA, Dalian, Liaoning, China) with the primers UL7-sense and UL7-antisense. The PCR products were purified using the Universal DNA Purification Kit (Tiangen, Beijing, China). Purified PCR products (400 ng) amplified from the genomic DNA extraction were re-annealed and treated with SURVEYOR nuclease (Transgenomics, Omaha, NE, U.S.A.). A nuclease capable of recognizing and digesting the previously mismatched nucleotides in the genome was used to confirm that the mutation in the UL7 gene was created by the CRISPR approach. The products were analyzed on 10 % TBE polyacrylamide gels, which were stained with ethidium bromide (EB) and imaged using a Bio-Rad Gel Doc gel imaging system. Quantification was based on the relative band intensities, as described by Cong et al. [24 (link)]. After detecting the mutation efficiency, mutated viruses were purified via plaque assay.
3
Lipid Metabolism Analysis: Detailed Reagents and Kits
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SYBR Premix Ex Taq (RR420A) and TRIzol Reagent (D9108B) were ordered from TaKaRa (Otsu, Shiga, Japan); a TIANamp Virus RNA/DNA Kit (DP315-R) was ordered from Tiangen (Beijing, China); a Dead Cell Apoptosis Kit with Annexin V-fluorescein (FITC) and PI (V13242), and BODIPY 493/503 (D3922) were ordered from Thermo Fisher Scientific (MA, USA); Hoechst 33342 (561908) was ordered from BD Biosciences (NJ, USA); Oil Red O (O0625) was ordered from Sigma-Aldrich (MO, USA); EGCG (HY-13653) was ordered from MedChemExpress (NJ, USA); a LabAssay nonessential fatty acid (294-63601) assay kit for FFAs was ordered from Wako Bioproducts (VA, USA); and a triglyceride assay kit (E1013) and a cholesterol assay kit (E1015) were ordered from Applygen Technologies Inc. (Beijing, China).
4
In vitro virus infection and quantification
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For in vitro virus infection, treated or untreated PK15 cells or HEK293 cells were washed 3 times with PBS and infected with PRV Bartha-K61 strain (MOI = 0.001) for 24 h or 48 h in a 37 °C, 5% CO2 incubator. After freeze-thawing of the cell suspension 3 times, the supernatant was collected by centrifugation. The DNA was extracted from the viral stock using TIANamp virus DNA/RNA Kit (TIANGEN, Beijing, China), and the copy number of PRV UL37 gene was detected by absolute RT-qPCR using the Premix Ex Taq Probe qPCR (Takara, Beijing, China); the viral stock was serially diluted 10 times, and the PRV titers were detected in BHK-21 cells by the Reed-Muench method. The primers and probe sequences of absolute RT-qPCR used were as follows: PRV-UL37-qF, 5′-GGACTACATGTTCCCCACGG-3′; PRV-UL37-qR, 5′-TAGAACGGCGTCAGGAATCG-3′; PRV probe, 5′-(FAM) CCACGGCCGTCACGA (Eclipse)-3′.
5
Viral DNA Extraction from Fecal and Serum Samples
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The fecal and serum samples were suspended at a proportion of 10% (wt/vol) in Dulbecco’s modified Eagle medium. The mixture was centrifuged at 8000×g at 4 °C for 20 min, and the supernatant was collected. Viral DNA was extracted using the TIANamp Virus DNA/RNA Kit (TianGen, Beijing, China) according to the manufacturer’s instructions and then stored at − 80 °C until analysis.
6
Sequencing and Annotation of Phage Genomes
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The propagated and purified phage stocks (109 PFU/mL) were centrifuged and filtered through 0.22 µm filter, then treated with 10 U/µL DNase I and RNase A (TakaRa, Dalian, China) and incubated at 37 °C for 1 h. The genomic DNA of the propagated phages was extracted using the TIANamp Virus DNA/RNA Kit (TIANGEN, Beijing, China) following the manufacturer’s instructions. The genomic DNA of phages 2L372D, 2L372X, 4L372X, 4L372XY, 44512, 44572, and 4D05 was sequenced at the Majorbio Cooperation (Shanghai, China) using Illumina HiSeq 2000. The resultant reads were assembled using SOAP denovo version 2.04 into a single contig for each phage [19 (link)]. The genomic DNA of phages 2D05 and 4L372D was sequenced using PacBio RS II. The resultant reads were assembled using SMRT Analysis version 2.3.0.
The genomes of nine phages were scanned for potential open reading frames (ORFs) with GeneMarkS (http://topaz.gatech.edu/GeneMark/ ) [20 (link)]. Annotation was carried out by comparing translated ORFs in BLASTP (http://blast.ncbi.nlm.nih.gov/Blast.cgi ) [21 (link),22 (link)]. tRNA genes were predicted by tRNAsan-SE version 1.21 [23 (link)].
The genomes of nine phages were scanned for potential open reading frames (ORFs) with GeneMarkS (
7
Detecting Deformed Wing Virus in Honey Bees
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Total RNA was extracted from the purified virus of China1-2017 and China2-2018 strains using a TIANamp Virus DNA/RNA Kit (Tiangen, Beijing, China), according to the manufacturer’s instructions. Total RNA was eluted in 30 μL of diethyl pyrocarbonate-treated water and stored at −80 °C until further analyses. To determine the presence of DWV in A. mellifera and A. cerana, a RT-PCR assay was performed using a PrimeScript™ RT-PCR Kit (TaKaRa, Dalian, China), according to the manufacturer’s instructions.
The primers DWV-F (5′-TTTGCAAGATGCTGTATGTGG-3′) and DWV-R (5′-GTCGTGCAGCTCGATAGGAT-3′) were used to amplify a 395 base pairs (bp) fragment of the DWV RdRp gene (accession number:AY292384 ). The PCR amplification was carried out under the following conditions: 94 °C for 2 min, followed by 25 cycles of 94 °C for 30 s, 55 °C for 30 s, 72 °C for 30 s, and a final extension of 72 °C for 5 min. A sample of six μL of PCR product was loaded on a 1.2% agarose gel containing Gelstain and analyzed using a Tanon 2500 Digital Gel Image Analysis System. The PCR products were sequenced commercially (Sangon Biotech, Shanghai, China).
The primers DWV-F (5′-TTTGCAAGATGCTGTATGTGG-3′) and DWV-R (5′-GTCGTGCAGCTCGATAGGAT-3′) were used to amplify a 395 base pairs (bp) fragment of the DWV RdRp gene (accession number:
8
Preparing Frozen Chicken Organ Samples and Viral Extracts
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Frozen chicken organ samples were homogenized for 15–30s before use. Cultured cell harvests were frozen and thawed several times followed by low speed centrifugation. The supernatants were stored at − 80 °C until use. Lyophilized vaccine samples were suspended in phosphate buffered saline following the manufacturer’s protocol. Viral genomic DNA and RNA were extracted from homogenates and lysed cell culture supernatants using TIANamp Virus DNA/RNA Kit following the company’s protocol (DP202, Tiangen Biotech, BeiJing). The samples after extraction were eluted in 70 μL RNase-free ddH2O and stored at − 80 °C.
9
DHBV cccDNA Purification Protocol
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DHBV total DNA in the culture medium was extracted using TIANamp Virus DNA/RNA kit (Tiangen Biotech Co., Ltd.). Following the removal of the culture medium, the cells were lysed and total DNA was extracted using TIANamp Genomic DNA kit (Tiangen Biotech Co., Ltd.). For the purification of DHBV cccDNA, DHBV total DNA was further treated with Plasmid-Safe™ ATP-Dependent DNase (Epicenter; Illumina, Inc., San Diego, CA, USA) at 37°C for 30 min followed by 70°C for 30 min to digest linear double-stranded DNA, and the resulting product was recycled using Cycle Pure kit (Omega Bio-tek, Inc.) according to the instructions of manufacturer.
10
Quantitative Detection of DHBV DNA
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For viral infection, virions of DHBV in the LMH D2 cell supernatants were precipitated with 10% PEG-8000 and dissolved in L-15 medium. The DHBV DNA in 200 μl L-15 medium was extracted with a TIANamp Virus DNA/RNA Kit (Tiangen, Beijing, China), followed by quantification using a TaqMan probe-based qPCR assay, which carried out with the primers and probe listed in Table 4 . The qPCR amplification was performed with the Takara Probe PCR Kit on an Eppendorf RealPlex 4, using 50 cycles of a 2-step PCR program.
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