type to induction conditions [6, 7], the technology of cloning, expression and purification of Taq polymerase in E.coli has been greatly improved. Nowadays, technologies in this area continue to develop.
Results and discussionsExtraction and purification of plasmid
Sample S and sample D were obtained through mini-prep isolation of plasmid pTaq1 fromE.coli culture prepared by students and demonstrator respectively. Sample S and D generated distinct concentration of pTaq1, which are respectively 19.3 ng/μl and 175.5 ng/μl (Fig.1a). Although D cutures produced significantly higher quantities of plasmid than S culture, both samples performed similar purity according to the 260/280 ratios. The 260/280 ratio of both samples were over 1.9 (Fig.1a), indicating that the plasmid samples can be considered free from organic contaminations but might contain RNA residue [7-9]. During the purification precedure, proteins and genomic DNA were neuralised and preciptated by the application of neutralisation buffer. Meanwhile, RNase in cell suspension buffer degrated RNA and reduced RNA residue but this action might have low efficiency and resulted in the relatively high value of 260/280. To improve the efficiency of RNA degration, RNase can be applied at a higher concentration or allowed longer reaction time.
Mini-prep isolation utilises alkaline extraction method, the main product of which is covalently closed circular (CCC) plasmid DNA due to its conformational stability under alkaline treatment [7, 10, 11]. However the 1kb DNA ladder consists of linear DNA fragments, which have lower migration rates than supercoiled plasmid DNA (CCC form) through agarose gel [10, 12, 13]. Therefore, The length of plasmid should be estimated greater than the corresponding value on the DNA ladder. According to the gel electrophoresis result, the length of purified plasmid DNA should be above 3000 bp as both samples had produced the brightest band at 3000bp level on the DNA ladder (Fig.1b). In sample D, a small amount of nucleic acids separated from supercoiled plasmid DNA (band3) was also observed (Fig.1b), which accorded with the diverse conformational forms of bacterial plasmid. Band1 indicated multimer forms of plasmid, which was formed by monomers interlocking together [7]. Multimer forms of plasmid have very slow migration rate because of their larger molecular size. We also discovered a band (band2) between multimers and supercoiled plasmid DNA (Fig.1b). This was likely to be the open circular (OC) form of plasmid, also known as nicked double-stranded DNA, which migrated slower than CCC plasmid DNA but faster than