DNA extraction from samples was performed using a Tissue DNA Extraction Kit (DP304; Tiangen Biotechnology Co., Ltd.) according to the protocol described by the manufacturer.
Tissue dna extraction kit
Manufactured by Tiangen Biotech
Sourced in China
The Tissue DNA Extraction Kit is a laboratory tool designed to isolate and purify genomic DNA from various tissue samples. It utilizes a fast and efficient process to extract high-quality DNA suitable for downstream applications such as PCR, sequencing, and genetic analysis.
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7 protocols using tissue dna extraction kit
1
Tissue DNA Extraction Protocol
2
Cochlear Gene Expression Analysis
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Six animals were sacrificed on day 1 after the third injection and six animals were sacrificed on day 3 after the ABR testing. The cochleae were dissected in Hanks’ balanced salt solution on ice and frozen in liquid nitrogen until use. Twelve cochleae (n = 4 for each group) of the animals sacrificed on day 1 were used for the extraction of total RNA. Twelve cochleae (n = 4 for each group) of the animals sacrificed on day 3 were used for genomic DNA (gDNA) extraction. The gDNA and total RNA were extracted using the Tissue DNA Extraction Kit (TIANGEN, DP304) and RNeasy Plus Mini Kit (QIAGEN, #74134) according to the manufacturer’s instructions, respectively. The purity and concentration of the DNA and RNA products were analyzed using the microplate spectrophotometer (BioTek, Epoch, USA). Then the isolated RNA was reverse transcribed to cDNA using PrimeScript RT Master Mix (TaKaRa, RR036A). The gDNA and cDNA products were stored at −20°C until use.
3
Quantitative Real-Time PCR for WSSV Detection
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Quantitative real-time PCR was performed to measure the WSSV copies in shrimp. The viral DNA was extracted from shrimp gills using a tissue DNA extraction kit (Tiangen, China), and the WSSV copies were detected by real-time PCR with WSSV-specific primers (5’-TTGGTTTCAGCCCGAGATT-3’ and 5’-CCTT GGTCAGCCCCTTGA-3’) and a WSSV-specific TaqMan probe (5’-FAM-TGCTGCCGTCTCCAA-Eclipse-3’) [27 (link)]. A linearized plasmid containing a 1400 bp DNA fragment from the WSSV genome was quantified and serially diluted 10-fold as an internal standard for real-time PCR. The 10 μl PCR solution contained 5 μl of Premix Ex Taq (Perfect Real Time) (TaKaRa, Japan), 0.2 μl of 10 μM forward primer, 0.2 μl of 10 μM reverse primer, 0.15 μl of 10 μM TaqMan fluorogenic probe, 200 ng of DNA template, and distilled water up to 10 μl. The real-time PCR conditions were 95°C for 1 min followed by 45 cycles of 30 s at 95°C, 30 s at 52°C, and 30 s at 72°C.
4
Quantitative WSSV Detection in Shrimp
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The WSSV copies of shrimp were determined by quantitative real-time PCR (qPCR). DNA extracted from shrimp hemocytes using a tissue DNA extraction kit (Tiangen) was subjected to qPCR with WSSV-specific primers (5′-TTGGTTTCAG CCCGAGATT-3′ and 5′-CCTTGGTCAGCCCCTTGA-3′) and probe (5′-FAM-TGC TGCCGTCTCCAA-Eclipse-3′). A linearized plasmid containing a 1400-bp DNA fragment from the WSSV genome was quantified and serially diluted as an internal standard. The 10 μl PCR solution contained 5 μl of Premix Ex Taq (Perfect Real Time) (TaKaRa), 0.2 μl of 10 μM forward and reverse primer respectively, 0.15 μl of 10 μM probe, 200 ng of DNA template, and distilled water up to 10 μl. The PCR program was 95°C for 1 min, followed by 45 cycles of 30 s at 95°C, 30 s at 52°C, and 30 s at 72°C.
5
Genotyping Transgenic Mice Expressing hACE2
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Genomic DNA was extracted from each mouse tail using a tissue DNA extraction kit (DP304, Tiangen) (Zeng et al., 2016 (link)). Genotyping was carried out using the following primers: hACE2-F: 5′-TGCTGGTTATTGTGCTGTCTCATCA-3′, and hACE2-R: 5′-CAGGTCTTCGGCTTCGTGGTTAA-3′. Polymerase chain reactions (PCR) were performed using Green Taq Mix (P131, Vazyme). PCR cycling conditions consisted of an initial activation step at 95°C for 15 min, followed by 30 cycles of denaturation at 95°C for 30 s, primer annealing at 58°C for 30 s, and extension at 72°C for 1 min, with a final extension step at 72°C for 7 min. PCR products were visualized on a 1% (w/v) agarose gel and the genotype of each animal was determined.
6
DNA Extraction from Paraffin Tissue
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DNA was extracted using a paraffin-embedded tissue DNA extraction kit (Tiangen Biochemical Technology Co., Ltd., DP331-02) according to instructions. The concentration and purity were detected by Onedrop OD-1000+ spectrophotometer detector.
7
Extracting DNA from Paraffin-Embedded Tissue
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Sample collection 10-15 pieces of embedded para n sections were taken and store at room temperature. DNA can be extracted directly with the para n-embedded tissue DNA extraction kit (Tiangen Biochemical Technology Co., Ltd., item number: DP331-02).
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