The CsF3′5′H gene from the NCBI database was subjected to standard end-to-end PCR reactions, with the primers designed according to the cDNA sequence (synthesized by Invitrogen, Shanghai, China; Additional file 2 : Table S1). The cDNA strands for end-to-end PCR were synthesized with Phusion® High-Fidelity DNA Polymerase (New England Biolabs, USA). PCR products were gel purified using the MiniBEST Agarose Gel Extraction Kit (Takara, DaLian, China), ligated into a pMD18-T vector, and transformed into E. coli DH5α competent cells for sequencing. The results were assembled using DNAMAN 7 software (Lynnon, Canada). Briefly, end-to-end PCR was performed under the following conditions: 98°C for 30 s, 30 cycles at 98°C for 30 s, 58°C for 10 s, 72°C for 40 s, and a final extension at 72°C for 10 min.
Minibest agarose gel extraction kit
Manufactured by Takara Bio
Sourced in China
The MiniBEST Agarose Gel Extraction Kit is a product designed for the extraction and purification of DNA fragments from agarose gels. It utilizes a simple and efficient spin-column method to recover DNA from gel slices.
Automatically generated - may contain errors
Lab products found in correlation
Phusion high fidelity dna polymerase, by New England Biolabs (3 mentions)
Rnaiso mate for plant tissue, by Takara Bio (2 mentions)
Rnaiso plus, by Takara Bio (2 mentions)
Primescript rt master mix, by Takara Bio (1 mentions)
Premix taq, by Takara Bio (1 mentions)
Trizol reagent, by Thermo Fisher Scientific (1 mentions)
Primescript 2 first strand cdna synthesis kit, by Takara Bio (1 mentions)
5 protocols using minibest agarose gel extraction kit
1
Cloning and Sequencing of CsF3'5'H Gene
2
Camellia sinensis RNA Extraction
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For analysis of gene expression, the leaves of Camellia sinensis var. sinensis. “Shuchazao” were collected for RNA extraction. The total RNA was isolated by RNAiso-mate for plant tissue (Takara, Dalian, China) and RNAiso Plus (Takara, Dalian, China). The cDNAs were amplified using Phusion® High-Fidelity DNA Polymerase (New England Biolabs, MA, USA), and the PCR products were purified via a MiniBEST agarose gel extraction kit (Takara, Dalian, China). The amplified PCR product was ligated into the pGEX-4T1 vector and transformed into TransT1-competent cells for sequencing.
3
Isolation and Cloning of Tea Plant Transcripts
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According to the manufacturer's instructions, total RNA was isolated using RNAisomate for Plant Tissue (Takara, Dalian, China) and RNAiso Plus (Takara, Dalian, China). The cDNA was synthesized by reverse transcription from the total RNA using PrimeScript RTMaster Mix (Takara, DaLian, China). The open reading frame sequence was amplified using Phusion High‐Fidelity DNA Polymerase (NewEngland Biolabs, MA, USA), and the PCR products were purified with a MiniBEST Agarose Gel Extraction Kit (Takara, Dalian, China) and ligated into the PGEX‐4T1 simple vector and subsequently transformed into TransT1 competent cells. Wild type tea plant tissues of different developmental stages (first leaf, second leaf, and third leaf), were harvested, and nucleic acid was isolated by the same method.
4
Construction of Recombinant TRV-based Vector
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The TRV2-GFP empty vector with EGFP-tagged coat protein (CP) fragment (Figure 7 ) exhibited similar efficiency in infection and gene silencing to TRV2 vector [51 (link)]; then, it was linearized using restriction enzymes (Xba I and Sac I) at 37 °C for 30 min in this study. The linearized vector was then linked with the isolated PlHB31 fragment using a Vazyme One Step Cloning Kit (Takara, Shiga, Japan), and the product was identified by agarose gel electrophoresis and purified using a Mini BEST Agarose Gel Extraction kit (Takara, Shiga, Japan). The purified recombinant plasmid was transformed into 100 μL of Escherichia. coli DH5α competent cells (Bioteke, Beijing, China) and then incubated in Luria–Bertani (LB) liquid medium for 1 h (37 °C, 200 rpm); thereafter, competent cells were selected on LBK plates containing 50 mg/L kanamycin after 12–16 h of incubation (37 °C, 200 rpm, in darkness). Monocolonies were inoculated into fresh LB medium, and plasmids were extracted for further DNA sequence alignment to verify the correct insertion of the fragment.
5
Glioma Cell Line Characterization by RT-PCR
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Cell culture. U251, A172 and U-87MG human glioma cell lines (Type Culture Collection of the Chinese Academy of Sciences, Shanghai, China) were maintained in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal bovine serum (FBS) (both from Gibco; Thermo Fisher Scientific, Inc., Waltham, MA, USA), 100 U/ml penicillin and 100 µg/ml streptomycin and were maintained at 37˚C and a 5% CO 2 humidified environment.
RNA extraction, reverse transcription-polymerase chain reaction (RT-PCR) and sequence alignment. Total RNA was isolated from cells using TRIzol reagent (Invitrogen; Thermo Fisher Scientific, Inc.), and cDNA was synthesized using PrimeScript II First-Strand cDNA Synthesis kit (Takara Bio, Inc., Otsu, Japan) at 42˚C for 60 min and 95˚C for 5 min. Subsequently, cDNA sequences were amplified by Premix Taq (Takara Bio, Inc.) with specific primers presented in Table I at 98˚C for 10 sec, 58˚C for 30 sec, 72˚C for 3 min and 72˚C for 5 min. DNA fragments were isolated from the agarose gels with Minibest Agarose Gel Extraction kit (Takara Bio, Inc.). PCR products were then subcloned into T-vector PMD19-T (Takara Bio, Inc.) and an individual clone was selected for sequencing by Sangon Biotech Co., Ltd., (Shanghai, China). Sequence alignments were performed with TCF4 genome (gene ID: 6934) deposited in Genbank by BLAST (blast.ncbi. nlm.nhi.gov/Blast.cgi).
RNA extraction, reverse transcription-polymerase chain reaction (RT-PCR) and sequence alignment. Total RNA was isolated from cells using TRIzol reagent (Invitrogen; Thermo Fisher Scientific, Inc.), and cDNA was synthesized using PrimeScript II First-Strand cDNA Synthesis kit (Takara Bio, Inc., Otsu, Japan) at 42˚C for 60 min and 95˚C for 5 min. Subsequently, cDNA sequences were amplified by Premix Taq (Takara Bio, Inc.) with specific primers presented in Table I at 98˚C for 10 sec, 58˚C for 30 sec, 72˚C for 3 min and 72˚C for 5 min. DNA fragments were isolated from the agarose gels with Minibest Agarose Gel Extraction kit (Takara Bio, Inc.). PCR products were then subcloned into T-vector PMD19-T (Takara Bio, Inc.) and an individual clone was selected for sequencing by Sangon Biotech Co., Ltd., (Shanghai, China). Sequence alignments were performed with TCF4 genome (gene ID: 6934) deposited in Genbank by BLAST (blast.ncbi. nlm.nhi.gov/Blast.cgi).
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