Protocols

Practical molecular biology methods from The Bond Lab. These summaries support education and research; validate reagents, times, and safety with your own lab standards before use. Shorter notes and updates also appear on Bond Lab Publications.

Lab protocols

Step-by-step outlines with buffers, key checkpoints, and sterile handling — open a card for the full page.

Pouring molten LB agar into a Petri dish for bacterial culture Pouring LB agar plates Sterilise LB agar, cool before antibiotics, pour under flame, dry, store, and QC selection plates for cloning. Infographic: cell fractionation by differential centrifugation — nuclei, mitochondria, microsomes, and cytosol Cell fractionation: nuclei extraction Hypotonic lysis and Dounce homogenisation to separate nuclear and cytoplasmic fractions for western blot. Infographic: non-viral cell transfection — plasmid DNA, DNA–lipid complex, uptake, endosome escape, and trans-gene expression Mammalian electroporation buffer (homemade recipe) 500 ml mammalian electroporation buffer from commodity reagents — sterile prep, cold storage, and cost versus small commercial vials. Infographic: Ras and Rap GST pull-down assays — RAF RBD and RalGDS RBD on agarose beads, GTP-bound pull-down, and representative western blots Ras/Rap activation assays (GST pull-down) GST-RBD bead prep, pull-down of active Ras or Rap from transfected cells, imaging and lysis buffers, and Kelley et al. RIPA-based Ras binding assay. Infographic: cell fractionation by differential centrifugation — nuclei, mitochondria, microsomes, and cytosol Quick F:G actin ratio sample prep Triton-soluble cytosol vs SDS-insoluble fractions for actin polymerisation and nuclear translocation western blots. Infographic: preparation of polyacrylamide hydrogels for cell culture — mixing, polymerization, equilibration, and seeding Collagen-coated polyacrylamide hydrogels on GelBond PAG film Cast soft (0.7 kPa) or stiff (50 kPa) PA gels, punch discs, and coat with collagen via sulfo-SANPAH for stiffness-dependent cell culture. Micrograph of 293 cells with calcium phosphate precipitate and DNA during transfection Calcium phosphate transfection of 293 cells CaPO₄ transfection in 15 cm dishes, 1× HBS recipe, and scaling DNA amounts for other formats. Infographic: regeneration and reuse of silica-based nucleic acid purification columns Regenerating silica-based DNA purification columns NaOH regeneration, RNase-free washes, and validation — cut consumable cost and plastic waste.

Use responsibly

Protocols here are not clinical SOPs. Follow institutional biosafety rules, waste streams, and any local requirements for GMO work, human samples, or radioactivity.