PCR reactions were run as total volume of 50 [micro]L reaction mixtures consisting of nuclease-free water, 5 [micro]L 10x PCR Buffer, 2 [micro]L 25 mM MgCl2, 1.5 [micro]L 10 mM deoxyribonucleotide
triphosphate mix (ABI, Applied Biosystems), 6 [micro]L of each primer (4 pmol/[micro]L), 0.25 [micro]L of Hot Start Taq DNA polymerase, and 200 ng of each tumor DNA.
The primers C (5'-CCTGCCTCGTATTAACTGCACCAT-3') and D (5'-CCGAGTGCTCCACATAGCCATGT-3') were used to amplify a 349-bp HP2 allele-specific sequence.25 Hp genotypes were determined by polymerase chain reaction (PCR) using 20uL total volume reactions containing 2 U/uL of Taq polymerase, 1-100 ng of DNA, and 200uM deoxyribonucleotide
triphosphatess (dNTPs) mix; PCR buffer was used as suggested by the supplier (Fermentas Co, Canada).
The apoptosis of endothelial cells in the tissue sections was detected by the terminal deoxyribonucleotide
transferase-mediated nick-end labeling (TUNEL) assay kit (Keygen, China), according to the manufacturer's instructions.
PCR was carried out in 30 [micro]l reaction mixture containinglOX buffer (100mM of TrisHCl, pH 8.3, 20mM Of MgCl2, 500mM of KCL and 0.1% gelatin) 200mM of deoxyribonucleotide
triphosphate (dATP, dTTP, dGTP and dCTP), 10 picomoles of each primer and 1 U of Taq polymerase (Bangalore Genei, Bangalore), with 2 [micro]l of template DNA.
Amplification was performed using 5-10 ng of fungal DNA as template in a 50 [micro]L mix containing 100 ng of each primer, 10 [micro]M of deoxyribonucleotide
triphosphate mixture, 2.5 mM of magnesium chloride, 5 [micro]L of 10X PCR buffer (200 mM Tris, pH 8.4; and 500 mM KCl) and 2.5 U of TaqDNA polymerase (Invitrogen, Carlsbad, CA, USA).
Cytoplasmic serine hydroxy-methyltransferase mediates competition between folate-dependent deoxyribonucleotide
and S-adenosylmethionine biosyntheses.
The real-time PCR was carried out in a mixture of 1 [micro]L of cDNA, 0.75 U of GoTaq DNA polymerase (Promega, USA), 5 [micro]L of 5xbuffer, 0.2 mM of deoxyribonucleotide
tri-phosphate (Promega, USA), 2.5 [micro]L of 3,000xSYBR Green I (BMA, Rockland, ME, USA), and 10 pmol of each primer.
triphosphate (dNTP) (Takara, Cat.
The final reaction volume was 35 [micro]L containing 10.2 [micro]L autoclaved Milli-Q water, 3.5 [micro]L deoxyribonucleotide
triphosphates (dNTP) (Life Technologies), 0.8 [micro]L Taq DNA polymerase (Biotools), 2.5 [micro]L MgCl2-free buffer (Biotools), 2.5 [micro]L MgCl2 (Biotools), 0.14 [micro]M of each 16S primer, 0.86 [micro]M of each MRS primer, 0.35 [micro]M of each SAU primer, 0.57 [micro]M of each COA primer, and 3.5 [micro]L of bacterial DNA.
DNA samples from each patient and control were amplified in a 50 [micro]L reaction mixture volume, containing 200 ng of DNA, 2.5 mmol/L magnesium chloride (MgC[l.sub.2]), deoxyribonucleotide
triphosphates (dNTP) mix (2.5 mmol/L each), oligonucleotide primers (10 pmol each), and Taq DNA polymerase (2.5 U/[micro]L).
The reactions were performed in a total volume of 25[micro]L containing 20ng of DNA, 1x buffer (10mM TrisHCl, 50mM KCl) (Phoneutria, Belo Horizonte, MG, Brazil), 5.5mM of Mg[Cl.sub.2] (Phoneutria), 200[micro]M of each deoxyribonucleotide
(dATP, dCTP, dGTP, and dTTP), 1.0[micro]M of each primer (Eurofins Scientific, Luxembourg), and 1.25 units of Taq DNA polymerase (Phoneutria).
All reactions contained ~1 unit Taq polymerase, 1x PCR buffer (50 mM KCl2, 10 mM Tris-HCl pH 9.0, 0.1% Triton X-100; Promega Corp., Madison, WI), 0.5 [micro]M deoxyribonucleotide
triphosphates, and 0.025 to 0.1 [micro]g DNA template.