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, gelatin and casein zymography, grant writing tips, and paper publication tips, and how to use B.E.E.P. (ENCODE promoter & ChIP overlap). 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

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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. 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.