Geneamp pcr 9700 thermal cycler

Oligonucleotide primers were designed using the MegAlign Clustal W program (DNASTAR Inc., Madison, WI) to obtain the whole genome of NVAV strain Te34 from cDNA prepared from total RNA, extracted from NVAV-infected Vero E6 cells and from lung tissue of the original wild-caught European mole (Table 2). The 5′– and 3′–termini of each segment were amplified using the 3′–Full RACE Core Set (Takara Bio Inc., Otsu, Japan). Nested or hemi-nested PCR was performed in 20 μL reaction mixtures, containing 250 μM dNTP, 2.5 mM MgCl₂, 1 U of Takara LA Taq polymerase (Takara) and 0.25 μM of each primer (Table 2). Initial denaturation at 94 °C for 2 min was followed by two cycles each of denaturation at 94 °C for 30 sec, two-degree step-down annealing from 46 °C to 38 °C for 40 sec, and elongation at 72 °C for 1 min, then 30 cycles of denaturation at 94 °C for 30 sec, annealing at 42 °C for 40 sec, and elongation at 72 °C for 1 min, in a GeneAmp PCR 9700 thermal cycler (Perkin-Elmer, Waltham, MA)34 (link). PCR products were separated, using MobiSpin S-400 spin columns (MoBiTec, Goettingen, Germany), and amplicons were sequenced directly using an ABI Prism 3130 Genetic Analyzer (Applied Biosystems, Foster City, CA)34 (link).

Bacteria were grown overnight on Columbia agar medium supplemented with 5% sheep blood. The bacterial DNA was prepared by QIAamp DNA Mini Kit (Qiagen Inc., Valencia, Calif., USA) according to the manufacturer’s instructions. The primer used for PCR was AP4 [30 (link)].
The reaction mixture contained 10 mM Tris-HCl (pH 8.3), 1.8 mM MgCl2, 2 μM primer, 400 μM each deoxynucleoside triphosphate, 2.5 U of Super-Therm Gold DNA polymerase (JMR Holdings), and 2μL of DNA in a final volume of 25μL. Amplification was performed in a GeneAmp PCR 9700 thermal cycler (Perkin-Elmer). Initial denaturation was for 4 min at 94°C. Amplification was performed with 50 cycles of 1 min at 94°C, 2 min at 30°C, and 2 min at 72°C, with a single final extension step of 10 min at 72°C. PCR products were separated by electrophoresis in a 1.5% agarose gel and stained with ethidium bromide.A CRKP strain, S, is included in all runs to ensure comparability between different runs.