multimers [10, 13]. Another minor band (band4) also appeared in Fig.1b. This band represented degraded RNA or RNA residue with low molecular weight [10, 13]. In comparison, plasmid sample purified from culture S produced only a single band of 3500bp DNA fragments, with relatively low intensity (Fig.1b). It can be explained that low bacterial concentration in culture S gave rise to low plasmid concentraition along with low quatities of other components that were not sufficient to be seen in gel electrophosis.
Taq gene purification
Samples were treated with restriction enzyme EcoRI, whose two sites on the pTaq1 plasmid flank the taq gene. EcoRI digestion yielded two bands, and the one measuring around 1800bp contained the taq gene (Fig.2a). The digestion of plasmid can be considered completed, since there were only two bands observed. Although the DNA sample contained diverse plasmid forms, they all resulted in identical fragments after the EcoRI digestion. EcoRI digestion produced linear double-stranded DNA fragments, thus the 1kb marker has accurate estimation to the length of these fragments. The size of pTaq1 can be estimated by adding together the length of band1 and band2, which was around 5000bp. Nevertheless, undigested pTaq1 (Fig.1b and Fig.2a) migrated appreciably faster than 5000bp linear fragments in the marker, on account of the particular high migration rate of supercoiled plasmid through agarose gel. The control groups presented similar band distribution to the plasmid in Fig.1a, suggesting that adjuvants (restriction buff and water) for digest process did not contaminate or affect the samples.
EcoRI digest was followed by gel purification to harvest the taq gene. Sample S yielded 5.7 ng/μl DNA with a 260/280 ratio of 2.42 and sample D yielded 9.1 ng/μl DNA with a 260/280 ratio of 2.12 (Fig.2c). A 260/280 ratio greater than 2 could be considered an indicator of high RNA concentration [7-9]. We conjectured that poor purity of DNA samples was caused by the RNA residue from plasmid samples. And the impact of RNA contamination on 260/280 ratio became more obvious when DNA concentration was very low. Gel purification of EcoRI digest product in sample D generated a small amount of 2000bp DNA fragment encoding the Taq polymerase, while sample S did not produce any visible band (Fig.2b), most likely due to the particularly low concentration of the product.
Expression of Taq DNA polymerase
Each colony was developed from a single bacterium successfully transformed with the plasmid.