following the standard procedures. Purified products were separated by electrophoresis in 1% TAE agarose gel containing Sybr Safe dye (InvitrongenTM S33102). The Sybr Safe dye stained the nucleic acids that fluoresced when exposed to UV light. 1kb plus DNA ladder (InvitrongenTM 10787-018) was used to identify the approximate molecular size of DNA. TE buffer and gel loading buffer were made up by students. All gel electrophoresis used the same chemicals in our experiment. Plasmid concentration and purity was determined from A260 and
A260/A280 ratio respectively, using a Nanodrop spectrophotometer.
Taq gene purification
To prepare the gene fragment encoding the Taq DNA polymerase, 10μl plasmid DNA was digested by 2μl restriction enzyme EcoRI (New England Biolabs R0101S, 10000units) and incubated at 37°C for 40 minutes. We also included a control reaction in which no EcoRI was added. The pTaq1 plasmid has two EcoRI sites flanking the taq gene and therefore can be digested into two linear DNA fragments. The fragment containing the taq gene has a smaller molecular weight and should migrate faster than the other fragment through agarose gel. After running the EcoRI digest product on an agarose gel we excised the fragment corresponding to the Taq polymerase gene and dissolved the gel slice in threefold volumes of buffer QG in 50°Cwaterbath for 10 minutes. Chemicals and column used to purify taq gene fragment came from the QIAQuick Gel Extraction kit (Qiagen 28704). The concentration of purified DNA was determined by gel electrophoresis as well as Nanodrop spectrophotometer.
Expression and purification of Taq polymerase
For protein expression, taq gene was transformed into E.coli cells using pETBlue-1 plasmid vector. The vector had been previously cleaved with the EcoRI restriction enzyme and treated with alkaline phosphatase to prevent re-ligation. The taq DNA fragments with complementary sticky ends [3] were ligated to plasmid vectors by T4 DNA ligase. The ligation reaction was assembled on ice and then incubated at room temperature (22°C) for 20 minutes. After ligation, the recombinant plasmids were transformed into E.coli cells following traditional Cacl2/heat shock transformation procedure. 3μl ligation products, 3μl supercoiled pETBlue-1 plasmid (positive control) and 3μl water (negative control) were added to every 50μl competent E.colicells, incubated on ice for 15 minutes, heat-shocked at 42°C for 90 seconds and then allow to cool on ice for 2 minutes. For each treatment, 200μl bacterial suspension was plated onto an