Recombinant taq dna polymerase

Specific regions of the gene encoding the 16S rRNA gene were amplified using the primers U968f GC and L1401r (Heuer and Smalla, 1997 ) in a solution containing 1X PCR buffer, 0.2 mM dNTPs, 2.5 mM MgCl₂, 2.5 U of recombinant Taq DNA polymerase (Promega, Madison, WI, USA), 10–40 ng of total DNA, 200 μmol of each primer and sterile Milli-Q water in a final volume of 50 μl. The reaction was performed in a thermocycler (Mastercycler Gradient, Eppendorf, Hamburg, Germany) with the following conditions: initial denaturing at 94°C for 3 min; 35 cycles at 94°C for 1 min, 55°C for 1 min and 72°C for 1 min; and a final extension at 72°C for 10 min. The denaturing gradient gel elecrtophoresis (DGGE) gels (45–65% urea and formamide) were prepared with a solution of polyacrylamide (6%) in Tris-acetate (pH 8.3). Electrophoresis was performed in Tris-acetate-EDTA buffer at 60°C at a constant voltage of 75 V for 16 h. The DGGE gels were stained with SYBR Green (Invitrogen, Carlsbad, CA, USA) and visualized using a Storm 860 Imaging System (GE Healthcare, Milwaukee, WI, USA).

Genomic ESC DNA was isolated using Blood & Tissue Kit (QIAGEN, Germany). For PCR reaction, recombinant Taq DNA polymerase (Promega) was used. The reaction was performed in 25 μl containing 1 μl DNA, 0.25 μl Go-Taq 2.5 μl PCR buffer (10X), 2 μl 1.5 mM MgCl₂, 0.5 μl of 10 mM dNTP, 0.3 μl of primer. PCR conditions for ESC analysis started with denaturation at 94 °C for 3 min followed by a primer-dependent number of cycles with denaturation at 94 °C for 30 s, annealing temperature at 61 °C for 45 s, and product elongation at 72 °C for 1 min. The following gene locus specific primer sequences have been used. To verify the recombination of the HPRT locus a so-called “lox-in” PCR was performed: LOXIN R 5′-ATA CTT TCT CGG CAG GAG CA-3′, and LOXIN F 5′-CTA GAT CTC GAA GGA TCT GGA G-3′. Genotyping of the PKD2-kinase-dead mice and iPSC lines was carried out by PCR of genomic DNA using primers 671–5′armF (5′-AGTGGCACGTTCCCCTTCAATG-3′) and 671–3′armR (5′-CTTTGCCCAATCCCTTACAGCCT-3′), producing products of 236 bp [PKD2WT (wild-type PKD2)] and 344 bp (PKD2-kinase-dead).