DNA fragments are separated based on size or molecular weight. Once placed in an electric field, fragments of small molecular weight move faster than large molecules (Asubel, Brent, Kingston, Moore, Seidman, Smith & Storuh 1995). This yields a distinctive banding pattern with each band representing a DNA fragment. The bands on agarose gels can be visualised through staining with DNA-staining dyes, such as ethidium bromide, followed by UV illumination (300-nm). In this practical, it was hypothesised that the band sizes will decrease from top to bottom of the gel because smaller fragments migrate further down the gel while large ones only move for short distances.
The blue/white screen is a method used to detect clones bearing a specific recombinant vector (a product of ligation) (Birnboim & Doly 2001). First, a gene of interest (Pc-ftsZ) is isolated from a donor cell and ligated into a vector (pBKS II). The recombinant vector is then used to transform competent recipient cells. The bacterial culture contains a substrate known as X-gal. Positive ‘tranformants’, i.e., bacteria transformed through successful ligation, will form white colonies while those that have not been transformed will remain blue. In this practical, since not all bacterial cells could be made competent through heat shock, it was expected that plating would give rise to both white and blue colonies. In this regard, only the E. coli cells from the white colony were selected for the PCR amplification step in practical four.
