HEK293T cells were seeded in 10 cm tissue culture plates and co-transfected with 10 μg pNL4.3/ΔVif and 0.25 to 10 μg pCMV4_A3G_HA wild type or mutant expression vectors using polyethylenimine (PEI, Molecular Biosciences). Total DNA levels were kept constant with empty pCMV4-HA vector. At 48 h, supernatants were harvested, DNAse (RQ1 RNase free DNAse (Promega)) treated for 1 h at 37°C and viruses then purified through a 20% (w/v) sucrose cushion at 28,000 x g for 75 min at 4°C. Viruses were resuspended in PBS, and quantified according to p24Gag content using an enzyme-linked immunosorbent assay (ELISA; Perkin-Elmer).
Dnase rq1 rnase free dnase
Manufactured by Promega
Sourced in United States
DNAse (RQ1 RNase free DNAse) is a lab equipment product that is used to remove DNA from RNA samples. It is an essential tool for researchers working with RNA-based experiments, as it helps to ensure the purity and integrity of RNA samples by eliminating any contaminating DNA.
Automatically generated - may contain errors
Lab products found in correlation
Revertaid first strand cdna synthesis kit, by Thermo Fisher Scientific (2 mentions)
Rneasy plant mini kit, by Qiagen (1 mentions)
Pgem t easy vector, by Promega (1 mentions)
Ckmix 2 ml, by Bertin Technologies (1 mentions)
Rneasy mini kit, by Qiagen (1 mentions)
Agilent 2100 bioanalyzer, by Agilent Technologies (1 mentions)
Quantinova sybr green pcr master mix, by Qiagen (1 mentions)
Cfx384 touch real time pcr detection system, by Bio-Rad (1 mentions)
5 protocols using dnase rq1 rnase free dnase
1
Quantification of HIV-1 Vif-Mediated A3G Restriction
2
RNA Editing Analysis in Pinus thunbergii
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Seedlings of the black pine P. thunbergii were grown under continuous light or continuous dark at 25 °C. Cotyledons were harvested at 10 and 14 days of age. RNA was extracted from cotyledons using RNeasy Plant Mini Kit (Qiagen) and DNase (RQ1 RNase-Free DNase; Promega) and was used for RT-PCR analyses and sub-cloning of chlB fragments. After preparation of cDNA from the extracted RNA, PCR was carried out using the gene-specific primers (Supplementary Table S2 ) with 25 cycles for chlB, chlL, psbA, rbcL, and rrn23 genes and 30 cycles for chlN and psbB genes. Amplified chlB fragments were cloned into pGEM-T Easy Vector (Promega), and nucleotide sequences were determined in both directions using the primer pair M13 forward and M13 reverse (3730XI, ABI). For each condition 33 or 36 clones were sequenced (Supplementary Table S1 ). RNA editing efficiency was defined as the number of edited clones/total number of clones.
3
Comprehensive RNA Extraction and RT-PCR
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Up to 30 mg of each tissue was homogenized in homogenization tubes (tissue grinding CKMix 2 ml, Bertin Technologies, Montigny-le-Bretonneux, France) in 100 µl of RNA lysis buffer (RNeasy
Mini Kit, Qiagen, Germany) at 5000 rpm for 3 × 30 sec (Minilys® personal homogenizer, Bertin Technologies). Total RNA was extracted using the RNeasy Mini Kit (Qiagen) and treated with DNase
(RQ1 RNase-Free DNase, Promega, USA). The Nanodrop ND-2000 (Wilmington, Delaware, USA) was used to assess the concentration and purity of isolated RNA. Additional control of RNA quality and
integrity was performed via microfluidic analysis using an Agilent 2100 Bioanalyzer (Agilent Genomics, USA); RNA integrity number (RIN) values were from 8.0 to 9.3 for the
domestic cat, 8.2 for the caracal, 8.6 for the Siberian tiger, and 5.4 for the clouded leopard. For RT-PCR, 1–2.5 µg of isolated RNA was reverse transcribed into single-stranded (ss)
complementary DNA (cDNA) using a RevertAid First Strand cDNA Synthesis Kit (Thermo Fisher Scientific, Waltham, MA, USA). Reverse transcriptase was not added to the negative control to verify
the absence of genomic DNA contamination.
Mini Kit, Qiagen, Germany) at 5000 rpm for 3 × 30 sec (Minilys® personal homogenizer, Bertin Technologies). Total RNA was extracted using the RNeasy Mini Kit (Qiagen) and treated with DNase
(RQ1 RNase-Free DNase, Promega, USA). The Nanodrop ND-2000 (Wilmington, Delaware, USA) was used to assess the concentration and purity of isolated RNA. Additional control of RNA quality and
integrity was performed via microfluidic analysis using an Agilent 2100 Bioanalyzer (Agilent Genomics, USA); RNA integrity number (RIN) values were from 8.0 to 9.3 for the
domestic cat, 8.2 for the caracal, 8.6 for the Siberian tiger, and 5.4 for the clouded leopard. For RT-PCR, 1–2.5 µg of isolated RNA was reverse transcribed into single-stranded (ss)
complementary DNA (cDNA) using a RevertAid First Strand cDNA Synthesis Kit (Thermo Fisher Scientific, Waltham, MA, USA). Reverse transcriptase was not added to the negative control to verify
the absence of genomic DNA contamination.
4
Bronchoalveolar Lavage and Gene Expression Analysis
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Three days following glucose tolerance testing (Group 2), mice were euthanized by an overdose i.p. injection of ketamine and xylazine and then tracheostomised for bronchoalveolar lavage (BAL) fluid cell counts [17 (link)]. The BAL fluid was centrifuged at 400 g for 4 min and the cell pellet was resuspended in phosphate-buffered solution. Lavage samples were processed for total BAL cell counts as previously described [18 (link)]; resuspended cells were identified using trypan blue staining and total cell counts were performed using a haemocytometer. Lungs and WAT (female, ovarian region; male, epididymal region) were dissected and weighed. Right lungs from mice at 6 weeks of age were snap frozen to analyse the expression of Kiss1. Homogenised lung tissue was treated with DNase (RQ1 RNase-free DNase, Promega) during RNA isolation to avoid genomic DNA contamination. Samples were analysed by quantitative real-time PCR (qPCR) using designer primers (Table 1 ) with QuantiNova SYBR Green PCR Master Mix (Qiagen), using CFX384 Touch Real-Time PCR Detection System (Bio-Rad). Standard curves were used to interpolate Ct values of target mRNA and normalised to reference genes: peptidylprolyl isomerase A (PPIA), succinate dehydrogenase (SDHA) and TATA-binding protein (TBP).
5
Quantification of HIV-1 Vif-Mediated A3G Restriction
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