Electrophoresis of DNA in Nondenaturing Polyacrylamide Gels

McGookin, R.

doi:10.1385/0-89603-127-6:75

Cited by 4 publications

(3 citation statements)

References 2 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…Polyacrylamide gels for DNA analysis were prepared according to McGookin (1988) with the 20× Gel Running Buffer containing 1 M Tris, 1 M boric acid, 200 mM EDTA, pH 8.3. Gel volume sufficient for two gel cassettes (15 ml) at 6% contained 0.75 ml of 20× Gel Running Buffer, 11.13 ml of water, 3 ml of 30% acrylamide/bis-acrylamide (29:1) (Bio-Rad, 1610156), 105 µl of 10% ammonium persulfate (Sigma-Aldrich, A3678), 15 µl of TEMED (Sigma-Aldrich, T9281).…”

Section: Methodsmentioning

confidence: 99%

A rapid and effective method for screening, sequencing and reporter verification of engineered frameshift mutations in zebrafish

Prykhozhij

Steele

Razaghi

et al. 2017

Disease Models &Amp; Mechanisms

View full text Add to dashboard Cite

Clustered regularly interspaced palindromic repeats (CRISPR)/Cas-based adaptive immunity against pathogens in bacteria has been adapted for genome editing and applied in zebrafish (Danio rerio) to generate frameshift mutations in protein-coding genes. Although there are methods to detect, quantify and sequence CRISPR/Cas9-induced mutations, identifying mutations in F1 heterozygous fish remains challenging. Additionally, sequencing a mutation and assuming that it causes a frameshift does not prove causality because of possible alternative translation start sites and potential effects of mutations on splicing. This problem is compounded by the relatively few antibodies available for zebrafish proteins, limiting validation at the protein level. To address these issues, we developed a detailed protocol to screen F1 mutation carriers, and clone and sequence identified mutations. In order to verify that mutations actually cause frameshifts, we created a fluorescent reporter system that can detect frameshift efficiency based on the cloning of wild-type and mutant cDNA fragments and their expression levels. As proof of principle, we applied this strategy to three CRISPR/Cas9-induced mutations in pycr1a, chd7 and hace1 genes. An insertion of seven nucleotides in pycr1a resulted in the first reported observation of exon skipping by CRISPR/Cas9-induced mutations in zebrafish. However, of these three mutant genes, the fluorescent reporter revealed effective frameshifting exclusively in the case of a two-nucleotide deletion in chd7, suggesting activity of alternative translation sites in the other two mutants even though pycr1a exon-skipping deletion is likely to be deleterious. This article provides a protocol for characterizing frameshift mutations in zebrafish, and highlights the importance of checking mutations at the mRNA level and verifying their effects on translation by fluorescent reporters when antibody detection of protein loss is not possible.

show abstract

Section: Methodsmentioning

confidence: 99%

A rapid and effective method for screening, sequencing and reporter verification of engineered frameshift mutations in zebrafish

Prykhozhij

Steele

Razaghi

et al. 2017

Disease Models &Amp; Mechanisms

View full text Add to dashboard Cite

show abstract

“… Cast two 8% non-denaturing TBE polyacrylamide mini gels. 4 Pool the PCR reactions and add 110 μL of 6× DNA loading dye. Load the PCR product across all available lanes.…”

Section: Step-by-step Methods Detailsmentioning

confidence: 99%

Optimized protocol for quantifying 5′ UTR-mediated translation initiation in S. cerevisiae using direct analysis of ribosome targeting

Lewis

Niederer²,

Neupane³

et al. 2022

STAR Protocols

View full text Add to dashboard Cite

“…RNA.Extract RNA from biological samples or transcribe in vitro. It is often preferable to purify RNAs prior to probing, i.e., via denaturing polyacrylamide gel electrophoresis [10] or using commercially available kits, but this is not always essential. Shorter RNAs (<400 nt) are generally best suited for the applications presented here and may be engineered to contain a 3′ structure cassette [4] to maximize the amount of native sequence that is readable.…”

Section: Methodsmentioning

confidence: 99%

Novel Biochemical Tools for Probing HIV RNA Structure

Rausch

Sztuba-Solińska

Lusvarghi

et al. 2016

Methods in Molecular Biology

View full text Add to dashboard Cite

Functional analysis of viral RNA requires knowledge of secondary structure arrangements and tertiary base interactions. Thus, high-throughput and comprehensive methods for assessing RNA structure are highly desirable. Selective 2'-hydroxyl acylation analyzed by primer extension (SHAPE) has proven highly useful for modeling the secondary structures of HIV and other retroviral RNAs in recent years. This technology is not without its limitations however, as SHAPE data can be severely compromised when the RNA under study is structurally heterogeneous. In addition, the method reveals little information regarding the three-dimensional (3D) organization of an RNA. This chapter outlines four detailed SHAPE-related methodologies that circumvent these limitations. "Ensemble" and "in-gel" variations of SHAPE permit structural analysis of individual conformers within structurally heterogeneous mixtures of RNA, while probing strategies that utilize "through-space" cleavage reagents such as methidiumpropyl-EDTA (MPE) and peptides appended with an ATCUN (amino terminal copper/nickel binding motif) can provide insight into 3D organization. Combinational application of these techniques provides a formidable arsenal for exploring the structures of HIV RNAs and associated nucleoprotein complexes.

show abstract

Electrophoresis of DNA in Nondenaturing Polyacrylamide Gels

Cited by 4 publications

References 2 publications

A rapid and effective method for screening, sequencing and reporter verification of engineered frameshift mutations in zebrafish

A rapid and effective method for screening, sequencing and reporter verification of engineered frameshift mutations in zebrafish

Optimized protocol for quantifying 5′ UTR-mediated translation initiation in S. cerevisiae using direct analysis of ribosome targeting

Novel Biochemical Tools for Probing HIV RNA Structure

Contact Info

Product

Resources

About