Q5 High-Fidelity 2X Master Mix was used for all PCR reactions in accordance to the manufacturer’s instructions. For all cloning procedures, NEBuilder HiFi DNA Assembly Master Mix was used in accordance to the manufacturer’s instructions. Escherichia coli strain DH5α was used for transformations and plasmid isolation. Synthesised gene fragments and custom oligonucleotide primers (Table 2 ) were ordered from Integrated DNA Technologies.
Custom oligonucleotide primers
Manufactured by Integrated DNA Technologies
Sourced in United States
Custom oligonucleotide primers are synthetic DNA sequences designed to serve as probes or starting points for DNA amplification or sequencing. They are used in various molecular biology applications, including polymerase chain reaction (PCR), DNA sequencing, and gene expression analysis.
Automatically generated - may contain errors
Lab products found in correlation
Gblocks gene fragment, by Integrated DNA Technologies (1 mentions)
Escherichia coli strain xl10 gold, by Agilent Technologies (1 mentions)
Stbl2 cells, by Thermo Fisher Scientific (1 mentions)
Perfecta sybr green supermix, by Quanta Biosciences (1 mentions)
Qscript supermix, by Quanta Biosciences (1 mentions)
Realplex2 qpcr system, by Eppendorf (1 mentions)
Trizol reagent, by Thermo Fisher Scientific (1 mentions)
N3011, by New England Biolabs (1 mentions)
Bacteriophage lambda dna, by New England Biolabs (1 mentions)
P4005, by Merck Group (1 mentions)
Nanodrop lite, by Thermo Fisher Scientific (1 mentions)
La taq dna polymerase, by Takara Bio (1 mentions)
Qiaquick gel extraction kit, by Qiagen (1 mentions)
7 protocols using custom oligonucleotide primers
1
Optimized Molecular Cloning Procedures
2
Generating PFBV and PEMV2 Mutants
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Plasmid pUC19-PFBV contains the full-length PFBV genome (NC_005286) downstream of a T7 promoter and was synthesized by GenScript (Piscataway, NJ). Plasmid pUC19-PEMV2 contains the full-length PEMV2 RNA2 genome downstream of a T7 promoter (26 (link)). PFBV and PEMV2 mutants were generated by QuikChange one-step site-directed mutagenesis (28 (link)) using custom oligonucleotide primers (Integrated DNA Technologies) (Supplemental Table S1 ). Resultant PCR products were subjected to DpnI digestion and transformed into chemically competent DH5α E. coli cells. Mutations were confirmed by sequencing (Eurofins Genomics). The JIN-PTE-WT fragment (D86123; positions 3738–3880) and JIN-PTEmPK fragment were purchased as gBlocks Gene Fragments from Integrated DNA Technologies.
3
Cloning and Tagging of p10 Constructs
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ARV p10, MdRV p10, and Md-CM (MdRV p10 containing the 9-residue conserved motif of ARV p10 created using sequential PCR reactions with Custom oligonucleotide primers) were subcloned into pcDNA3 mammalian expression vectors between the HindIII and EcoRI sites. A triple FLAG or single Myc tag was added to the N-terminus, or EGFP or mCherry were added to the C-terminus of the indicated p10 constructs by PCR amplification and cloning. Custom oligonucleotide primers were purchased from Integrated DNA Technologies (IDT), and all constructs were confirmed by sequencing.
4
Amplification and Transcription of PFBV PTE
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PFBV PTE and JIN-PTE fragments were PCR amplified using custom oligonucleotide primers (Integrated DNA Technologies) (all oligonucleotide primer sequences are available upon request). Forward primers contained an upstream T7 promoter. For in vitro translation trans-inhibition assays, the PFBV PTE fragment amplified was positions 3619–3781, and for SHAPE structure probing, the PFBV PTE fragment amplified was positions 3619–3792 and the probe annealing site 5′GGTCATAGCTGTTTCCT was added to the 3′end. Resulting PCR products were used as templates for in vitro transcription using T7 RNA polymerase.
5
Preparation and Use of HIV-1 Env Plasmids
6
Quantifying Gene Expression in Cell Cultures
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Total RNA was isolated from cultures using TriZol reagent (Life Technologies, Carlsbad, CA, USA) according to the manufacturers recommended instructions. cDNA was reverse transcribed from 1 µg total RNA using qScript SuperMix (Quanta Biosciences, Gaithersburg, MD, USA) according to manufacturers instructions. qPCR was carried out using a 5 ng equivalent of cDNA in a 1X reaction of PerfeCTa SYBR Green SuperMix (Quanta Biosciences, Beverly, MA, USA) and 250 nM each (forward and reverse) custom oligonucleotide primers (Integrated DNA Technologies, Coralville, IA, USA) generated by using PrimerBlast (National Center for Biotechnology Information, Bethesda, MD, USA). Reactions were carried out on an Eppendorf RealPlex2 qPCR system, and fold changes in gene expression were calculated using the ΔΔCT method using species-specific GAPDH primers. Primer sequences are listed in Table S1 . Human collagen 1 and α-SMA expression levels in the 2D FibCon condition were used as the reference group for fibroblast gene expression, and mouse myogenin gene expression levels of 2D MyoCon were used as a reference for myoblasts. N = 5 for all groups.
7
Synthesizing DNA Fragments Using PCR
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We generated DNA of lengths 217 nm (639 bp), 398 nm (1170 bp), and 1023 nm (3008 bp) using PCR (36) . Specifically, we used bacteriophage lambda DNA (New England Biolabs N3011; Ipswich, MA) as a template, custom oligonucleotide primers (Integrated DNA Technologies; Coralville, IA), and an LA Taq DNA polymerase (TaKaRa Bio RR004; Kusatsu, Japan). We verified that products had amplified correctly using gel electophoresis and then extracted the DNA using a commercial kit (Qiagen QIAquick Gel Extraction Kit, 28704; Hilden, Germany). Finally, we measured the concentration and purity using a spectrophotometer (ThermoFisher NanoDrop Lite; Waltham, MA). Samples with A260/A280 ratios of less than 1.7 were discarded.
We purchased protamine from salmon (Sigma-Aldrich P4005; Saint Louis, MO), diluted it in deionized water, and stored 30 M aliquots at -20 C.
We purchased protamine from salmon (Sigma-Aldrich P4005; Saint Louis, MO), diluted it in deionized water, and stored 30 M aliquots at -20 C.
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