Etoposide was from Sigma and actinomycin D from Millipore. The V5-tagged A3A, A3C, A3F, A3G and A3H cDNAs in the pcDNA3.1D/V5-His-TOPO expression vector (Invitrogen) have been described15 (link). All constructs were grown in E. coli DH5α. A3C siRNA (HSS120676, HSS120678, HSS178439), A3A siRNA (HSS153372, HSS153373, HSS153374) and siRNA negative control are from Thermoscientific.
Pcdna3.1d v5 his topo expression vector
Manufactured by Thermo Fisher Scientific
Sourced in Japan
The PcDNA3.1D/V5-His-TOPO expression vector is a plasmid used for expression of recombinant proteins in mammalian cell lines. It features a CMV promoter for high-level expression, a V5 epitope tag, and a polyhistidine (6xHis) tag for detection and purification of the expressed protein.
Automatically generated - may contain errors
5 protocols using pcdna3.1d v5 his topo expression vector
1
Evaluating APOBEC3 protein function
2
HIV-1 env Gene Cloning for Expression
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Full length HIV-1 env genes were re-amplified from the first-round SGA products as previously described [41 (link)]. The PCR product was cloned into the pcDNA3.1D/V5-His-TOPO expression vector (Invitrogen) using the pcDNA 3.1 directional TOPO expression kit (Invitrogen).
3
Plasmid Cloning of Mas and MrgD Receptors
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Plasmids for the human Mas (GenBank accession no. M13150.1) and human MrgD (GenBank accession no. AY427820.1) receptors were obtained from Kazusa DNA Research Institute (Chiba, Japan).
The expression vectors were subcloned from their respective cDNAs by PCR using the following primers: for the human Mas receptor,5′-CACCATGGATGGGTCAAACGTGACATCATTT-3′ (forward) and 5′-AAAGACGACAGTCTCAACTGTGACCGTATT-3′ (reverse; lacking a stop codon); for the human MrgD receptor, 5′-CACCATGAACCAGACTTTGAATAGCAGTGGG-3′ (forward) and 5′-AAAAGCCCCCATCTCATTGGTGCCCACGGT-3′ (reverse; lacking a stop codon).
The PCR products were cloned into the pcDNA3.1D/V5HisTOPO expression vector (Invitrogen) and subsequently sequenced. Protein expression was confirmed by western blotting in transfected differentiated 3T3L-1 adipocytes.
The expression vectors were subcloned from their respective cDNAs by PCR using the following primers: for the human Mas receptor,
The PCR products were cloned into the pcDNA3.1D/V5HisTOPO expression vector (Invitrogen) and subsequently sequenced. Protein expression was confirmed by western blotting in transfected differentiated 3T3L-1 adipocytes.
4
Constructing Env Expression Plasmids
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Several expression plasmids were generated from env genes of 4 cluster individuals (LTNP_1, LTNP_3, LTNP_RF_21, and AS7), 4 HIV-1 chronic progressors (I_10, IV_10, I_14, and RIS_06), and 3 laboratory-adapted viruses (SF162, NL4.3, and 89ES_061). The R5-tropic BaL.01-env (catalog no. 11445) glycoprotein plasmid was from the NIH AIDS Research and Reference Reagent Program. The env genes were amplified by nested PCR from proviral DNA (35 (link)). The products were cloned into the pcDNA3.1D/V5-His Topo expression vector (Invitrogen). In total, 9 clones derived from the 4 cluster patients (LTNP_1 clones 8, 10, 12, and 37; LTNP_3 clones 20, 22 and 3; LTNP_RF clone 21; and AS7), 6 clones from chronic progressors (I_10 clones 27 and 28, IV_10 clones 5 and 16, RIS_06 clone 2, and patient I_14 clone 9), and 4 clones from reference samples (SF162, 89ES_061, NL4.3, and BaL.01) were constructed. pcDNA3 plasmids coding for NL4.3-derived mutants D368R and 41.2, which abrogate CD4 binding and gp41-mediated membrane fusion, respectively, were generated by site-directed mutagenesis or by subcloning from original plasmids, as previously described (24 (link)). Expression plasmids were transformed in DH5α cells, and clones were sequenced to check the correct insertion of the env gene.
5
Cloning and Characterizing env Genes
About PubCompare
Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.
We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.
However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.
Ready to get started?
Sign up for free.
Registration takes 20 seconds.
Available from any computer
No download required
Revolutionizing how scientists
search and build protocols!