Developed in his workshop at home, where he had made one-off toy trucks for his own sons from sheets of tin, Hornby's breakthrough came in designing a kit of perforated components.
One other limitation of recombination-based systems is that they require the purchase of commercial kits or kit components, which can represent a significant cost when working with large gene sets.
Total protein normalization was not possible due to the presence in the luciferase kit lysis buffer of components that disrupt common total protein measurement assays.
Each reaction mixture (20 μL) contained an amount of RT product equivalent to 100 ng of input RNA, 0.25 μmol/L each PCR primer (Table 1) and standard amounts of kit components in the fast SYBR green master mix.
Conditioned media and lysed cells were spun at 250 g for 15 min at 4°C followed by addition of kit components, incubated for 30 min and read at 490 nm.
The assembly kit consisted of eight groups of components, plus a carry case.
Peptides able to bind to dsDNA, and therefore immobilized in the microtitre surface, were detected by streptavidin horseradish peroxidase conjugate (KPL, Gaithersburg, MD, USA) at a concentration of 0.5 μg/ml in combination with 3,3',5,5'-tetramethylbenzidine 3,3',5,5'-tetramethylbenzidineISA).
Binding of SmD-derived peptide to dsDNA was studied using dsDNA-coated ELISA plates (kit component of dsDNA ELISA, catalogue number 25004; Dr. Fooke Laboratories).
Unbound peptides were removed by three washing cycles with 350 μl washing buffer (kit component of the dsDNA ELISA) per well.
