Preliminary RT-LAMP assay development and optimisation was undertaken using cloned RNA transcripts of the complete HIV-1 integrase (IN) coding region. The IN coding region of the 8E5 strain of HIV-1 (clade B) was amplified by nested PCR using primers 5′-CTCACAGTATGCATTAGGAATYAT and 5′-CCTTATGGCAGATTCTGAAAAACA in the first round and 5′-GCACACAAAGGAATTGGAGGAAAT and 5′-TAGTGGGATGTGTACTTCTGAACT in the second round. The PCR products were cloned into the pCR 2.1-TOPO Vector (Life Technologies, Paisley, UK). DNA was extracted using a Plasmid Minikit (Qiagen, Manchester, UK) and the plasmid linearized by digestion with restriction enzyme Pst1 (New England Biolabs, Hitchin, UK). RNA transcripts were made using the MEGAScript T7 polymerase kit (Life Technologies) according to the manufacturer’s instructions. Post-transcription, the nucleic acid was DNAse digested (DNAse 1 amplification grade, Life Technologies) for 30 minutes at 37 °C and RNA extracted with the QIAamp viral RNA kit (Qiagen).
Restriction enzyme pst1
Manufactured by New England Biolabs
Sourced in United Kingdom
Pst1 is a type II restriction enzyme that recognizes and cleaves the palindromic DNA sequence 5'-CTGCAG-3'. It is commonly used in molecular biology applications for DNA digestion and fragment analysis.
Automatically generated - may contain errors
Lab products found in correlation
Dnase 1 amplification grade, by Thermo Fisher Scientific (1 mentions)
Pcr2.1 topo vector, by Thermo Fisher Scientific (1 mentions)
Plasmid mini kit, by Qiagen (1 mentions)
Qiaamp viral rna kit, by Qiagen (1 mentions)
Inos antibody, by Cell Signaling Technology (1 mentions)
H3k27me3 antibody, by Merck Group (1 mentions)
Recombinant mouse gm csf and il 4, by Thermo Fisher Scientific (1 mentions)
2 protocols using restriction enzyme pst1
1
Cloning and Transcription of HIV-1 Integrase
2
Mouse Macrophage Polarization Assay
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Male and female C57BL/6 mice (6‐8 weeks) were obtained from Joint Ventures Sipper BK Experimental Animal (Shanghai, China). No randomization of weight and sex or blinding was used for animal studies. Dicer1d/d mice (6‐8 weeks) were kindly gifted by Dr. Jiahuai Han (Xiamen University, Xiamen, Fujian, China) [14 ]. All animal experiments were performed following the National Institute of Health Guide for the Care and Use of Laboratory Animals, with the approval of the Scientific Investigation Board of Second Military Medical University (SMMU, Shanghai, China). Lipopolysaccharides (LPS) (0111:B4) (Sigma‐Aldrich, St. Louis, Missouri USA) stimulation was performed following previously described methods [15 ]. Recombinant mouse GM‐CSF and IL‐4 were obtained from PeproTech (London, UK); restriction enzyme Pst1 from NEB (Ipswich, Massachusetts, USA); H3K27me3 antibody from Millipore (Burlington, Massachusetts, USA); Mi‐2β antibody from Santa Cruz (Santa Cruz, California, USA); iNOS antibody from Cell Signaling Technology (Beverly, Massachusetts, USA).
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