RNA were extracted using Trizol reagent (TransGen Biotech, Beijing, China) as described by the manufacturer. The RT-PCR Access kit (Promega, Wisconsin, USA) was used to detect the transcription of F and NA genes. The method was essentially the same as that described previously using primer pairs #1 or #2 (Table 1 ) [3] (link). To include the pol(A) at 3′ end of expressed genes, the PCR products of NA gene (1431bp), and F gene (1692bp), were cloned directly into the pcDNA3.1(-) expressing vector (Invitrogen, California, USA) to form a new vector named as pcDNA-NA or pcDNA-F, and sequences were confirmed by automated sequencing (BGI, Beijing, China).
Pcdna3.1 expressing vector
Manufactured by Thermo Fisher Scientific
Sourced in United States
The PcDNA3.1(-) expressing vector is a plasmid DNA construct designed for the expression of recombinant proteins in mammalian cells. It contains a cytomegalovirus (CMV) promoter for high-level expression and a neomycin resistance gene for selection of transfected cells.
Automatically generated - may contain errors
Lab products found in correlation
Lipofectamine 3000, by Thermo Fisher Scientific (2 mentions)
Lipofectamine rnaimax, by Thermo Fisher Scientific (2 mentions)
Access rt pcr kit, by Promega (1 mentions)
Trizol reagent, by Transgene (1 mentions)
Freestyle 293 expression system, by Thermo Fisher Scientific (1 mentions)
Mouse mab isotyping kit, by Merck Group (1 mentions)
Sh nc, by GenePharma (1 mentions)
Lv shnc, by Genechem (1 mentions)
Lentiviral vector, by Genechem (1 mentions)
Plko 1 puro vector, by Addgene (1 mentions)
5 protocols using pcdna3.1 expressing vector
1
Extraction and Cloning of Viral Genes
2
Monoclonal Antibody Generation Against ErbB2
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The extracellular domain of ErbB2 (ErbB2-ECD) was prepared as described previously,15 (link) except that we used the pcDNA3.1(+)-expressing vector (Invitrogen, Waltham, MA, USA) and the FreeStyle 293 expression system (Invitrogen). Female BALB/c mice were repeatedly immunized with recombinant human ErbB2-ECD protein. Three days after the final immunization, spleens were collected and the splenocytes were fused to NS-1 mouse myeloma cells. The fused cells were cultured in hypoxanthine/aminopterin/thymidine medium. Culture supernatants from the resulting hybridomas were tested by ELISA for specific antibody reactivity to ErbB2-ECD. Antibody isotypes were determined by using a mouse mAb isotyping kit (Sigma, St Louis, MO, USA). Finally, the mouse anti-ErbB2 mAbs were purified by protein G affinity chromatography from hybridoma culture supernatants.
3
Overexpression and Silencing of UBE2L3 and LINC01116 in Prostate Cancer
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The full-length cDNA sequence of UBE2L3 was acquired and inserted into pcDNA3.1 expressing vector (Invitrogen) for overexpressing UBE2L3, with empty vector as control. The specific shRNAs to LINC01116 (sh-LINC01116#1/2) or UBE2L3 (sh-UBE2L3#1/2) and their relative negative control (sh-NC) were constructed by GenePharma (Shanghai, China). The miR-744-5p mimics and NC mimics were constructed by Ribobio (Guangzhou, China), as well as miR-744-5p inhibitor and NC inhibitor. All these were transfected for 48 h into DU145 and LNCAP, by means of Lipofectamine 3000 (Invitrogen). Each independent experiment was carried out at least thrice.
4
FOXK1 Expression Modulation Protocol
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The full-length FOXK1 cDNA (GenBank accession number NM_001037165) was amplified and subcloned to into pCDNA3.1 expressing vector (Invitrogen, Carlsbad, CA). The expression level of FOXK1 was then examined by western blot. Empty vector- transfected cells were used as a control.
Small interfering RNA (siRNA) targeting FOXK1 and negative control was specifically synthesized by GenePharma (Shanghai, China). Lipofectamine RNAiMAX (Thermo Fisher Scientific, Waltham, MA) was used to transfect siRNAs into cells according to the manufacturer's protocol. The siRNA sequences were as follows: siFOXK1 sense strand, 5′-GCAUGGGCCUGUCCAGCUUT−3′; the scrambled (src) siRNA 5′-TTCTCCGAACGTGTCACGT-3. The siRNA-transfected cells were harvested in the medium for 2 days after transfection and then used for subsequent studies.
Lentiviral vectors containing human FOXK1 short-hairpin RNA (Lv-shFOXK1) and scrambled non-targeting shRNA (Lv-shNC) which was served as negative control were provided by Genechem Company (Shanghai, China). The guiding strand sequences of Lv-shRNA were as follows: shFOXK1, 5′- CCTCTCTCTTAACCGCTACTT-3′; shNC, 5′- TTCTCCGAACGTGTCACGT-3′; Cells were infected with indicated lentivirus at an multiplicity of infection (MOI) of 20 for 48 h and then selected with puromycin (1.5 μg/mL) for 7 days. The expression level of FOXK1 in the infected cells was validated by western blot analysis.
Small interfering RNA (siRNA) targeting FOXK1 and negative control was specifically synthesized by GenePharma (Shanghai, China). Lipofectamine RNAiMAX (Thermo Fisher Scientific, Waltham, MA) was used to transfect siRNAs into cells according to the manufacturer's protocol. The siRNA sequences were as follows: siFOXK1 sense strand, 5′-GCAUGGGCCUGUCCAGCUUT−3′; the scrambled (src) siRNA 5′-TTCTCCGAACGTGTCACGT-3. The siRNA-transfected cells were harvested in the medium for 2 days after transfection and then used for subsequent studies.
Lentiviral vectors containing human FOXK1 short-hairpin RNA (Lv-shFOXK1) and scrambled non-targeting shRNA (Lv-shNC) which was served as negative control were provided by Genechem Company (Shanghai, China). The guiding strand sequences of Lv-shRNA were as follows: shFOXK1, 5′- CCTCTCTCTTAACCGCTACTT-3′; shNC, 5′- TTCTCCGAACGTGTCACGT-3′; Cells were infected with indicated lentivirus at an multiplicity of infection (MOI) of 20 for 48 h and then selected with puromycin (1.5 μg/mL) for 7 days. The expression level of FOXK1 in the infected cells was validated by western blot analysis.
5
Engineered shRNA and cDNA Constructs
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The shRNA oligonucleotides against H19 were synthesized by GenePharma (Shanghai, China) and cloned into the pLKO.1 puro vector (Addgene, USA). ATG7 cDNA was acquired through RT-PCR and then cloned into the pcDNA 3.1-expressing vector (Invitrogen). The transfection of the indicated constructs was conducted by using Lipofectamine 3000 following the manufacturer's guidance (Invitrogen, USA). After 48 h, the cells were used for subsequent experiments.
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