dna ladder  (New England Biolabs)

Journal: Nucleic Acids Research

Article Title: Template nicking suppresses promoter-independent antisense transcription in IVT via R-loop-mediated strand displacement

doi: 10.1093/nar/gkaf1536

Figure Lengend Snippet: Promoter-independent antisense transcription contributes to dsRNA. ( A ) 1% agarose gel electrophoresis analysis of eGFP RNA synthesized by T7 RNA polymerase and treated with increasing concentrations of S1 nuclease (0.04, 0.08, 0.16, and 0.24 U) or RNase III (0.002, 0.004, 0.008, and 0.016 U). ( B ) 1% agarose gel and J2 antibody-based dot blot analysis of RNA transcribed from four DNA templates: (i) ssDNA containing only the T7 promoter region (80 nt), (ii) single-stranded template strand (904 nt), (iii) partially duplexed DNA with 80 bp encompassing the T7 promoter and a single-stranded downstream region, and (iv) fully duplexed DNA. For both analyses, 200 ng of each IVT RNA was loaded. As controls, 200 ng of ssRNA and 20 ng of dsRNA markers were included on the agarose gel, and 200 ng of ssRNA and 10 ng of dsRNA were spotted on the dot blot to confirm J2 antibody specificity. Total RNA was quantified using the RiboGreen assay. Values for total RNA and dsRNA were normalized to the yield obtained from the fully duplexed dsDNA template. Statistical comparisons were performed using one-way ANOVA with Šídák’s multiple comparisons test; **** P < .0001.

Article Snippet: Strand specificity was validated by digesting 200 ng of ssDNA and dsDNA with 5 U S1 nuclease in 1 × S1 buffer or 5 U BsrGI-HF® (New England Biolabs, #R3575S) in NEB rCutSmartTM Buffer at 37°C for 20 min. Products were heat-inactivated at 80°C for 20 min and analyzed by 1% agarose gel electrophoresis (GelRed-stained) alongside the 100 bp DNA Ladder (New England Biolabs, #N0467S).

Techniques: Agarose Gel Electrophoresis, Synthesized, Dot Blot

Journal: Nucleic Acids Research

Article Title: Template nicking suppresses promoter-independent antisense transcription in IVT via R-loop-mediated strand displacement

doi: 10.1093/nar/gkaf1536

Figure Lengend Snippet: NiLoT suppresses dsRNA across different template formats, transcript sizes, T7 RNAP variants, and modified NTPs. ( A ) 1% agarose gel and J2 antibody-based dot blot analysis of RNA transcribed using either PCR-amplified or linearized plasmid DNA templates, prepared as dsDNA or NiLoT. 200 ng of each IVT RNA was loaded for both analyses. For reference, 200 ng of ssRNA marker and 20 ng of dsRNA marker were loaded on the agarose gel. Total RNA was quantified using the RiboGreen assay. Values for total RNA and dsRNA were normalized to the yield obtained from the PCR-based dsDNA template with unmodified NTPs. Statistical comparisons were performed using two-tailed, unpaired t -test; ** P < .01. ( B ) Agarose gel electrophoresis and J2 dot blot analysis of IVT products from 1.0 kb and 3.9 kb templates. RNAs were synthesized using either dsDNA or NiLoT templates encoding 1.0 kb (eGFP) or 3.9 kb (SARS-CoV-2 spike) transcripts. For each condition, 200 ng of IVT RNA was analyzed by 1% agarose gel electrophoresis and by dot blot using the J2 antibody. For reference, 200 ng of ssRNA and 20 ng of dsRNA were loaded on the gel as controls. Total RNA concentrations were measured using the RiboGreen assay, and dsRNA levels were quantified from dot blots. All values were normalized to the total RNA and dsRNA yield obtained from the corresponding dsDNA-derived transcripts. ( C ) Representative 1% agarose gel and J2 antibody-based dot blot analyses of IVT RNA transcribed from either conventional dsDNA or NiLoT templates using three T7 RNA polymerases: WT #1 (vendor 1, wild-type), WT #2 (vendor 2, wild-type), and Mutant #1 (vendor 1, engineered low-dsRNA variant). For each condition, 200 ng of RNA was loaded per assay. Reference lanes include 200 ng ssRNA and 20 ng dsRNA markers (gel), and 200 ng ssRNA and 10 ng dsRNA controls (dot blot). Total RNA was quantified using the RiboGreen assay. RNA yield and dsRNA content were normalized to the output from the dsDNA template transcribed with WT #1. Statistical comparisons were performed using a two-tailed, unpaired t -test; ** P < .01 and **** P < .0001. ( D ) Chemical structures and abbreviations of modified nucleotides used for IVT: Ψ (pseudouridine), m 1 Ψ (N1-methylpseudouridine), m 5 C (5-methylcytidine), and m 6 A (N6-methyladenosine). ( E ) 1% agarose gel and J2 antibody–based dot blot analysis of eGFP mRNA transcribed using either dsDNA or NiLoT templates, with or without modified NTPs shown in panel ( D ). 200 ng of IVT mRNA was loaded per lane/spot. For reference, 200 ng of ssRNA marker and 20 ng of dsRNA marker were loaded on the agarose gel, and 200 ng of ssRNA and 10 ng of dsRNA controls were included on the dot blot. The J2 blot confirms that NiLoT-derived mRNAs contain markedly lower levels of dsRNA contaminants than dsDNA-derived counterparts. Total RNA was quantified using the RiboGreen assay. Values for total RNA and dsRNA were normalized to the yield obtained from the dsDNA template with unmodified NTPs.

Article Snippet: Strand specificity was validated by digesting 200 ng of ssDNA and dsDNA with 5 U S1 nuclease in 1 × S1 buffer or 5 U BsrGI-HF® (New England Biolabs, #R3575S) in NEB rCutSmartTM Buffer at 37°C for 20 min. Products were heat-inactivated at 80°C for 20 min and analyzed by 1% agarose gel electrophoresis (GelRed-stained) alongside the 100 bp DNA Ladder (New England Biolabs, #N0467S).

Techniques: Modification, Agarose Gel Electrophoresis, Dot Blot, Amplification, Plasmid Preparation, Marker, Two Tailed Test, Synthesized, Derivative Assay, Mutagenesis, Variant Assay