Methods and guides

Free molecular biology methods and guides from The Bond Lab: qPCR and PCR primer design, western blotting buffers, western blotting tips, EMSA tips, melt curve analysis, qPCR data analysis, MIQE checklist for qPCR publications, GSEA interpretation guide, reporter gene assays (11-part guide), gelatin and casein zymography, grant writing tips, and paper publication tips, and how to use B.E.E.P. (ENCODE promoter & ChIP overlap), and mapping transcription factor binding sites. These explainers support education and research; confirm methods with your lab standards. See also protocols (e.g. calcium phosphate transfection) and free molecular biology tools (e.g. restriction enzyme digest map).

Guides

Open a card for the full article.

Reporter gene assays guide

An 11-part comprehensive guide on designing and running reporter gene assays — from reporter choice and vector design through controls, troubleshooting, and high-throughput screening. Start with Part 1.

DNA double helix with promoter and antibiotic-resistance gene labels Part 1 Introduction to reporter assays What reporter assays measure and why assay design matters as much as reporter choice. Part 2 Types of reporter genes Colorimetric, fluorescent, and luciferase reporters compared. Part 3 Secreted or cellular reporters When to sample medium versus lysing cells for repeated readouts. Part 4 Destabilised reporters PEST tags and short half-life reporters for kinetic measurements. Part 5 Choosing the right promoter Constitutive, inducible, and synthetic promoters for reporters. Part 6 Choosing the right vector Backbone elements, selection markers, and vector artefacts. Part 7 Assay normalisation Dual-reporter ratios and correcting for transfection efficiency. Part 8 Controls Positive, negative, promoterless, and empty-vector controls. Part 9 Troubleshooting Low signal, high background, and common failure modes. Part 10 High-throughput screening Z′, pilot plates, hit rates, and counter-screens. Part 11 Top 15 tips A practical checklist distilled from the full series.
Eukaryotic gene promoter diagram with transcription factors bound upstream of the transcription start site A practical guide to mapping and interrogating transcription factor binding sites From promoter motif scanning and ENCODE ChIP-seq to reporter assays, EMSA, ChIP, and ChEA3 — a complete TFBS workflow with BondLab tools. Antibody-shaped transcription factor binding a DNA double helix — B.E.E.P. promoter and ChIP analysis How to use the BondLab ENCODE Enabled Promoter (B.E.E.P.) analysis tools User guide: batch human promoter BED from RefSeq TSS, ENCODE TF ChIP peaks, bigWig signal overlap, boolean TF filters, and exports for IGV. Labeled western blot buffer bottles: PBS, TBST, TBS, SDS sample buffer, and transfer buffer Western blot lysis buffer recipe Lysis buffer for western blotting: why SDS often beats RIPA, with 50 ml SDS lysis and Super Western blocking buffer recipes. Western blot workflow graphic for gel transfer and antibody detection Top 10 tips for successful western blotting Practical western blotting advice: primary antibodies, transfer, blocking, ECL, reprobing, washing, and publication-quality blots from the Bond Lab. EMSA gel showing STAF/ZNF143 binding with unlabelled probe competition controls Top 10 tips for successful EMSAs Gel shift assay advice from the Bond Lab: protein quality, probe design, competitors, native PAGE, controls, and publication-quality EMSA data. Infographic on qPCR primer design goals, melting temperature, specificity, and best practices qPCR and PCR primer design Use NCBI Gene and Primer-BLAST to design human GAPDH primers — settings for product size, Tm, and specificity, plus tips on cost versus commercial assay kits. Melt curve plot showing two sample groups with distinct melting temperature peaks Melt curve analysis (qPCR) Melt analysis for SYBR Green qPCR: single vs multiple peaks, primer dimers, and how to read melt curves with amplification plots and controls. Casein zymogram with molecular weight markers and protease inhibitor lanes Gelatin and casein zymography Detect MMP-2, MMP-9, and MMP-3 by substrate-embedded gels: sample handling, renaturation, inhibitor controls, and interpreting clear bands. Laptop showing qPCR amplification curves and fold-change analysis in a laboratory qPCR data analysis Making sense of Ct values: QC, normalization, ΔΔCt, Pfaffl efficiency, fold changes, statistics, and MIQE reporting. MIQE qPCR guidelines card with DNA helix and amplification curve graphic MIQE checklist for qPCR publications A practical MIQE guide: RNA quality, primer validation, PCR efficiency, reference genes, controls, transparent methods reporting, and reproducibility. GSEA enrichment plot on a laboratory monitor showing ranked gene set analysis How to interpret GSEA results Gene set enrichment analysis explained: ranked lists, enrichment plots, ES, NES, FDR, leading edge genes, and common interpretation mistakes. Infographic: ten tips for successful grant application writing Grant writing tips Ten practical grant writing tips: timelines, the rule of three, preliminary data, value for money, and publishing.
Scientist in a lab coat reviewing a research manuscript with a red pen Paper publication tips Ten tips for getting your scientific paper published: journal choice, structure, figures, reviewers, and persistence.

More guides will be added over time. Suggestions are welcome via Contact Us.

About the author: This page was written by Dr Mark Bond from The Bond Lab at the University of Bristol. These notes reflect the methodology used in our cardiovascular and cell-signalling research. Questions about these methods: contact us or email mark.bond@bristol.ac.uk ORCID.