Accutaq la dna polymerase kit

The PCR was carried out for qualitative analysis of CEPBA and c-MYC expression according to the manufacturer's protocol for the AccuTaq™LA DNA Polymerase kit (Sigma Aldrich; Merck KGaA, Darmstadt, Germany). The reaction mixture contained: 0.7 μl of 10 μM of each primer (CEBPA and c-MYC respectively), 3.5 μl of 1.5 mM 10x PCR buffer without MgCl₂ (Sigma Aldrich; Merck KgaA), 0.7 μl of 25 mM MgCl₂, 0.4 μl of 0.2 mM dNTP (deoxynucleotides) mix, 0.2 μl of 0.5 U AccuTaq LA DNA Polymerase, 1 μl of cDNA and 13.8 μl distilled water. The final volume of reaction mixture was 21 μl. For each experiment a negative control without cDNA template was included. The PCR amplifications for investigated and reference genes were carried out using an MJ Mini Personal Thermal Cycler (BioRad Laboratories, Inc., Hercules, CA, USA). Thermal conditions were as follows: initial denaturation at 95°C for 2 min, denaturation at 95°C for 1 min, annealing at 56°C for CEBPA and 57°C for MYC for 30 sec, elongation at 72°C for 45 sec and final elongation at 72°C for 5 min. For visualization of PCR product, electrophoresis on a 2% agarose gels were used.

For qualitative analysis of the mRNA expression of the reference gene GAPDH, a PCR assay was performed in accordance with the AccuTaq™ LA DNA Polymerase Kit protocol (Sigma Aldrich, Germany). The mixture for PCR consisted of 10× AccuTaq buffer, 1 mM of MgCl₂, 0.5 μM of each primer (GAPDH gene: F 5′-TGGTATCGTGGAAGGACTCATGAC-3′, R 5′-ATGCCAGTGAGCTTCCCGTTCAGC-3′), 0.2 mM of each dNTP, 0.5U of AccuTaq LA DNA Polymerase, 0.2 μg of cDNA template and distilled water to a final volume of 20 μL. In every experiment, a negative control was included. Products of the PCR reactions were assessed by electrophoresis in 2 % agarose gel. The reaction product for GAPDH had a size of 257 bp.

For the studied polymorphism, polymerase chain reaction (PCR) was performed in accordance with the AccuTaq™ LA DNA Polymerase Kit protocol (Sigma Aldrich, Germany). The mixture for PCR consisted of 10× AccuTaq buffer, 1.5 mM of MgCl₂, 0.5 μM of each primer (C421A F 5′-ATGTTGTGATGGGCACTCTG-3′; C421A R 5′-TGCTGATCATGATGCTTTCAG-3′), 0.2 mM of each dNTP, 0.5U of AccuTaq LA DNA Polymerase, 50 ng of DNA template and distilled water to a final volume of 20 μL. In every experiment, a negative control was included. Products of the PCR reactions were assessed by electrophoresis in 2 % agarose gel. Reaction products for the investigated SNP had a size of 184 bp.