Ribonucleotide reductase (RR; EC 126.96.36.199) converts ribonucleoside diphosphates into deoxyribonucleoside
diphosphates being essential for DNA replication, which is why the enzyme is considered an excellent target for anticancer therapy.
The PCR reaction was performed in a thermocycler (GenePro, Bioer Technology, Hangzhou, China) at a total volume of 25 containing 2.5 [micro]L of 10X Taq buffer (1X) (Invitrogen, Carlsbad, CA, USA), 0.5 [micro]L of dNTP mix (25 [micro]M of each deoxyribonucleoside
triphosphate - dATP, dCTP, dGTP and dTTP) (Invitrogen, Carlsbad, CA, USA), 1.25 [micro]L of 25 mm Mg[Cl.sub.2], 0.25 [micro]L of forward and reverse universal primers (0.2 [micro]M) (Invitrogen, Carlsbad, CA, USA), 1.5 [micro]L sample DNA, 1.5 [micro]L Taq DNA polymerase (1 unit) (Invitrogen, Carlsbad, CA, USA), and 17.25 [micro]L nuclease-free water.
Toward single molecule DNA sequencing: direct identification of ribonucleoside and deoxyribonucleoside
5'-monophosphates by using an engineered protein nanopore equipped with a molecular adapter.
The primary amplification was performed by using the oligonucleotide primers PfATPase6P1 and PfATPase6-P2 as described elsewhere (4) in a reaction mixture of total volume 20 [micro]l which consisted of 5 pl of genomic DNA, 4 [micro]l of 5 x colourless Gotaq reaction buffer, 0.3 [micro]M (0.6 [micro]l in 20 [micro]l of reaction volume) of each primer, 0.2 mM (0.165 [micro]l in 20 [micro]l of reaction volume) of each deoxyribonucleoside
triphosphate (dATP, dTTP, dGTP, dCTP) and 1.2 units of taq DNA polymerase (0.24 [micro]l in 20 [micro]l of reaction volume).
The 50-L PCR mixture contained 8 L of DNA template 1 L (100 pmol) of each primer and a 25 L of Taq PCR Master Mix polymerase containing 100 mM Tris-HCl 500 mM KCl (pH 8.3) 1.5 mM MgCl2 200 M of each deoxyribonucleoside
triphosphate and 0.025 U of Taq polymerase (Qiagen USA).
The total volume (25 [micro]L) of the PCR reaction mixture contained 2.5 [micro]L of 10 x Taq buffer, 1.5 [micro]L of Mg[Cl.sub.2] (25 mmol [L.sup.-1]), 2 [micro]L of each deoxyribonucleoside
triphosphate (10 mmol L-1), 1 [micro]L of each primer (30 pmol), 1 [micro]L of DNA template, 0.5 [micro]L of Taq DNA polymerase, and 15.5 [micro]L of [H.sub.2]O.
Each 25 [micro]L PCR mixture contained 1 [micro]L (1:10 dilution) community DNA (10 ng-20 ng), 2.5 [micro]L PCR buffer (1X), 1 [micro]L of each deoxyribonucleoside
triphosphate (dNTP) (100 mM), 1 [micro]L of forward and reverse primers (0.5 [micro]M), and 0.5 [micro]L Taq polymerase (3U) (Fermentas).
A., Madrid, Spain), and 0.5 mM of deoxyribonucleoside
triphosphate mix (Roche, Basel, Switzerland).
In PCR mixture total volume of 50ul contained 2.0mM MgCl2,10mM Tris-HCl pH 8.0, 200uM of deoxyribonucleoside
triphosphate (dNTPs), 100 pmoles of each forward and reverse primer, 2.5 units of Taq DNA polymerase and 100-150 ng of crude DNA.
PCR mixtures consisted of PCR buffer with 1.5 mM of Mg[Cl.sub.2], 10 pmol of each primer, 2.0 [micro]M of each deoxyribonucleoside
triphosphate, 0.1 [micro]l of Taq DNA polymerase, and 1 [micro]l of template DNA solution in a final volume of 25 [micro]l.
The reaction mixture consisted of 30 uL of sterile water; 5 [micro]L of 10X Taq Buffer (100 mM Tris-HCl [pH 8.8 at 25[degrees]C], 500 mM KCl, 0.8% Non-idet P40); 1.5 [micro]L of 25 mM Mg[Cl.sub.2]; 1 [micro]L of deoxyribonucleoside
triphosphates (2 mM each dATP, dCTP, dTTP, and dGTP); 0.5 [micro]L of each primer (stock concentration, 40 UM); 10 [micro]L of template; and 0.2 [micro]L (5 U/[micro]l) of TaqDNA polymerase.
The PCR samples contained 10x PCR buffer, 2mM deoxyribonucleoside
triphosphate, 0.1 mM primers, and Taq polymerase (Applies Biosystems, Foster City, CA, USA).