Most of the bacteria harbour recombinant plasmid but some are transformed with empty vectors (Table.1). The low concentration of tag gene in both samples (Fig.2c) failed to meet the 1:3 molar ratio of vector to insert, which accounted for the incomplete plasmid recombination. By comparing the result of sample D and sample S, we found that higher concentration of the insert DNA contributed to a higher recombination frequency (Fig.2c and Table.1). Ligation of taq DNA fragment to vector plasmid produced a transformation efficiency of 5.17×104 cfu/μg in sample S and 6.97×104 cfu/μg in sample D, which were acceptable but relatively low [7]. In comparison, positive control transformed with supercoiled plasmid showed a higher transformation efficiency of 8.32×104 cfu/μg. According to previous researches [7, 14], transformation efficiency is compromised by the restriction cleavage of vector plasmid and religation of cleaved vector has little or no recovery of the reduced transformation efficiency. During the heat shock phase, the temperature of waterbath fluctuated to around 47°C, which might have killed some bacteria and led to the overall low efficiency. Some researches have reported that increasing plasmid concentration along with vigorous shaking could contribute to higher transformation efficiency [14].
The recombinant Taq polymerase was purified from induced E.coli (pTaq2) cells that contained the taq gene. Culture containing control plasmid without the taq gene (pControl) was also included. The expression of Taq DNA polymerase was demonstrated by comparing the SDS-PAGE of the proteins present in cell lysate of E.coli (pTaq2) to those present in cell lysate of E.coli (pControl) (Fig.3). Accordingly, we confirmed Taq polymerase expression inE.coli (pTaq2) and the molecular weight of this enzyme was around 92 kDa. However, we also observed a slight amount of Taq polymerase in the control samples, which was caused by sample contamination from next lane leakage, since we loaded the sample too early before running.
PCR
Subsequently, we examined purified Taq enzyme activity through PCR performance. PCR reactions were setup with commercial Taq polymerase or with Taq polymerase purified fromE.coli (pTaq2). Template DNA was successfully amplified using either Taq enzyme, showing an 1100 bp DNA fragment in 1% agarose gels. Moreover, two individual PCRs have produced very similar band distribution and intensity, suggesting that Taq polymerase purified in lab had nearly identical enzyme activity to commercial Taq polymerase (Fig.4). E.coli (pControl) cell lysate obtained through a parallel purification process was used to setup PCR reaction and no