( PCR Amplification ...)Amplification of the amoA Gene of Nitrosococcus oceani Bacteria Using ,,, Eppendorf MasterTaq

PCR Amplification
Two bacterial primers, A189, GGN GAC TGG GAC TCC TGG (Holmes et. al) and AmoA2-R, CCC CTC KGS AAA GCC TTC TTC (Rotthauwe et. al), were utilized to amplify a 675 bp region of the amoA gene of the cultures. PCR was performed as follows: 2 l of each primer (10 M), 1 l of N. oceani culture, 2.5 l of 10X buffer, 2 l of 2.5 mM dNTP, 2.5 U of Taq polymerase, and 0-5 l of 5X TaqMaster. This mixture was adjusted to a final volume of 25 l using
sterile water. A negative control (no DNA) was also amplified. Amplification of the target gene was carried out via the following program: 94 C for 2 minutes, 35 cycles of 94 C for 1 minute, 56 C for 1 minute, and 72 C for 1 minute, and a final extension step of 56 C for 5 minutes.

Visualization
PCR products were run in a 2.0% agarose gel and stained with ethidium bromide for visualization.

Results and Discussion

The 675 bp region of the amoA gene was successfully amplified using the Eppendorf MasterTaq polymerase kit. Varying the amount of TaqMaster thermostabilizer included in the PCR reaction had a minimal effect on the band obtained from N. oceani. The addition of 2.5 l of TaqMaster gave the most favorable results (Lane 4), though bands were visible at all concentrations of TaqMaster (Lanes 3 and 5). The additional bands seen in the N. oceani lanes are most likely the amo subunits. Internal probing after PCR verifies that the band seen at approximately 675 bp is indeed amoA (data not shown). Eppendorfs MasterTaq polymerase kit is a beneficial enzyme system to use when attempting to study known laboratory cultures of ammonia-oxidizing bacteria
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