TRIzol (Invitrogen, Carlsbad, CA, United States) was used to extract total RNA from the peripheral blood of healthy family members. First strand cDNA was synthesized using the HiScript II 1st Strand cDNA Synthesis Kit (Vazyme, Jiangsu, China). Primers containing EcoRI and SmaI (forward primer: 5′-ggaattctATGGCTACGCCCCTGGTGGC-3′, reverse primer: 5′-tcccccgggTCACTCCTTTTGCTCTGTGG-3′) sites were designed to amplify the coding sequence of the TINF2 gene. The PCR products were cloned into the pEGFP-C1 vector (GENEWIZ, New Jersey, United States) through double digestion with EcoRI and SmaI (New England Biolabs, Beijing, China). The mutant pEGFP-C1-TINF2-MUT vector was obtained by PCR-based site-directed mutagenesis using PrimerStar (Takara, Dalian, China). The primers were as follows: forward primer: 5′-GGTGTGGACATGGGTGGCTGCTTCCAGAG-3′ and reverse primer: 5′-CTCTGGAAGCAGCCACCCATGTCCACACC-3’. The constructs were sequenced to confirm that no secondary mutation was introduced. Plasmids were extracted using Endofree Plasmid Midi Kit (Tiangen, Beijing, China).
Endofree midi plasmid kit
Manufactured by Tiangen Biotech
Sourced in China
The EndoFree midi Plasmid Kit is a laboratory product designed for the purification of plasmid DNA. It utilizes a silica-based adsorption method to efficiently capture and purify plasmid DNA from bacterial cultures.
Automatically generated - may contain errors
Lab products found in correlation
Primerstar, by Takara Bio (1 mentions)
Pegfp c1 vector, by Genewiz (1 mentions)
Trizol, by Thermo Fisher Scientific (1 mentions)
Ecori, by New England Biolabs (1 mentions)
Hiscript 2 1st strand cdna synthesis kit, by Vazyme (1 mentions)
Pcdna3.1 myc his a, by Thermo Fisher Scientific (1 mentions)
Geneticin g418, by Selleck Chemicals (1 mentions)
E coli dh5α competent cells, by Sangon (1 mentions)
Lipofectamine 3000, by Thermo Fisher Scientific (1 mentions)
Puromycin, by Yeasen (1 mentions)
5 protocols using endofree midi plasmid kit
1
TINF2 Gene Cloning and Mutagenesis
2
Construction and Validation of BFNNV Capsid Plasmids
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Based on the sequence of the open reading frame (ORF) of BFNNV capsid (GenBank accession NO. KM576685), primers with restriction sites were designed (Table 1 ). Based on the abundance of antigen sites, RNA2 was divided into two fragments, namely, the N-terminal fragment (1–414 nt) and the C-terminal fragment (415–1014 nt), respectively. The N-terminal, C-terminal, and the whole RNA2 fragments were inserted into the empty plasmid pEGFP-N1, and named pEGFP-CP-N, pEGFP-CP-C and pEGFP-CP, respectively. The sequence of pEGFP-CP, pEGFP-CP-N and pEGFP-CP-C was confirmed by restriction enzyme digestions and DNA sequencing. The ORF of BFNNV capsid was cloned into the eukaryotic expression vector pcDNA3.1-myc-His A (Invitrogen, Waltham, MA, USA) using EcoR Ⅰ and Hind Ⅲ restriction enzyme sites to obtain the plasmid pcDNA3.1-CP, which will be used for a potential plasmid DNA vaccine in T. rubripes. The plasmids pEGFP-CP, pEGFP-CP-N, pEGFP-CP-C and pcDNA3.1-CP were purified using the Endo-free Plasmid Midi Kit (Tiangen Biotech, Beijing, China), and then stored at −20 °C until used (Figure 1 ).
3
Lentiviral shRNA Knockdown of ALPK2 Gene
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The RNA interference target sequence (5′-GCGAAGACCTTGGCATTTATT-3′) was designed based on the RNA interference sequence design principle for ALPK2. Lentiviral shRNA vector targeting ALPK2 was constructed by Shanghai bioscienceres Co. Ltd. (Shanghai, China). The sequences targeting of ALPK2 were synthesized and cloned into a lentiviral vector BRV-108 (Shanghai Yibeirui bioscienceres Co. Ltd., Shanghai, China). Monoclonal clones on the plate were selected for PCR identification, and the positive clones were sequenced and analyzed. The positive clones were cultured and extracted to obtain high purity plasmids (EndoFree midi Plasmid Kit, TIANGEN, Cat. #DP118-2) for downstream virus packaging. 293T cells were co-transfected with three plasmids (BRV-108, Helper 1.0 and Helper 2.0) to obtain lentivirus. The negative control group was transfected with negative lentivirus shCtrl, and the shALPK2 group was transfected with shALPK2. After 72 h, the expression of green fluorescent protein was observed with a fluorescence microscope to evaluate the transfection efficiency.
4
Lentiviral Overexpression and Knockdown of Key Proteins
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Flag-CRIP1, empty vector and shRNA plasmids were purchased from Youbio Biological Technology (Changsha, China). The full-length coding sequence of CRIP1 was inserted into pCDH-EF1-MCS-T2A-Puro. shRNAs targeting CRIP1, PSME4 and USP7 were purchased from OBiO Technology (Shanghai, China). Endotoxin-free transfection-grade plasmid DNA was amplified in E. coli DH5α Competent Cells (Sangon Biotech, Shanghai, China) and purified with the EndoFree Midi Plasmid Kit (TIANGEN, Beijing, China). Lentiviruses were constructed using psPAX2 and pMD2G and transfected into HEK293T cells with Lipofectamine 3000 (Invitrogen, USA), harvested after 48 or 72 h, and concentrated through ultracentrifugation (20,000×g, 2.5 h, 4 °C). The lentivirus was added to 1 106 cells along with 8 mg/mL polybrene in a 12-well plate, and stable cells were selected after 72 h using puromycin (60210es25, Yeasen, China) and geneticin (G418; Selleck, USA). The levels of CRIP1 were confirmed using RT-qPCR and western blotting. shRNA sequences are listed as follows: CRIP1-shRNA (5′-AGCACGAAGGCAAACCCTACT-3′); PSME4-shRNA (5′-GCAACTAGTAAATCTCTTTGC-3′); and USP7-shRNA (5′-GTGTCCTATATCCAGTGTAAA-3′).
5
Engineered Lentiviral Silencing of CDC42EP3
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Three target sequences of CDC42EP3 were designed as follow: 5′- CGGACTCTGTGTTCACAGAAA-3′, 5′-AAGCTCTCATGTTGCCCTTAT-3′, 5′- ATGCGAGCTCATCAAGGGAAA-3′. shRNAs targeting CDC42EP3 silence based on the above sequences were further designed and prepared by Shanghai Bioscienceres Co., Ltd. (Shanghai, China). Then the shRNA sequences were cloned into BR-V-108 linearized vector using Fermentas T4 DNA Ligase (Thermo Fisher Scientific). Verified lentiviruses were transfected into Top 10 E.coli receptor cells for amplifying and high purity plasmid (EndoFree midi Plasmid Kit, TIANGEN) were extracted. 293 T cells were used for packaging and lentiviral harvest was performed 72 h after transfection.
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