The reaction was done in a final volume of 20 ul containing 2 ul of 10 X PCR buffer without magnesium chloride(MgCl2) (Fermentas, Lithuania), 1 ul of 25 mM MgCl2 (Fermentas, Lithuan4a), 1 ul 2mM deoxyribonucleotide triphosphate
(dNTPs) (Fermentas, Lithuania), 0.1 ul of 5U Taq DNA polymerase enzyme (Fermentas, Lithuania), 1 ul of 20 uM of forward and reverse primers, 2 ul of 40 ng/ul human genomic DNA and volume of the reaction was made up to 20ul with autoclaved deionised water.
 Nonstandard abbreviations: qPCR, quantitative PCR; Cq, quantitation cycle; HRM, high-resolution melting; dNTP, deoxyribonucleotide triphosphate
PCR amplifications were carried out in 25 [micro]L reaction mixtures containing 2 mM mixture of deoxyribonucleotide triphosphate
phate (dNTP), 2.0 [M[g.sup.2+]], 1 mM of forward and reverse primers.
Two microliters (1 [micro]L for nested PCR) DNA were amplified in a 50-[micro]L mixture containing 10 pmol of each primer, 200 [micro]mol/L each deoxyribonucleotide triphosphate
(Invitrogen, Cergy-Pontoise, France), 1.5 U Taq polymerase (Invitrogen), and 2.5 [micro]L of a 50-mmol/L solution of Mg[Cl.sub.2] in 1x Taq buffer.
Prewarmed reaction buffer containing DTT (dithiothreitol, 5 mM final) and dNTPs (deoxyribonucleotide triphosphate mix, 0.5 mM final) was added.
Abbreviations: DNTP, deoxyribonucleotide triphosphate mix; DTI', dithiothreitol; EDTA, ethylenediaminetetraacetic acid; EST, expressed sequence tag; GMGI, Glycine max gene index; Mbp, megabasepairs; MW, molecular weight; NEG, negative; PCR, polymerase chain reaction; RT, reverse transcriptase; TC, tentative consensus; TIGR, The Institute for Genomic Research.
Reaction mix includes deoxyribonucleotide triphosphate
(dNTP) and 2'-deoxy-iso-guanosine triphosphate (iso-dGTP) modified with a fluorescent quencher.
A ready-to-use 2xPCR mixture from AandA Biotechnology (Poland) was used, comprised of a recombinant Taq DNA polymerase (0.1 U/l), PCR buffer optimized by the manufacturer, magnesium chloride (4.0 mM), a mixture of deoxyribonucleotide triphosphates
(0.5 mM of each dNTP), a red dye, gel loading buffer (enabling direct application of the reaction mixture on an agarose gel) and deionized water.
(1,2) The Sanger method is also known as terminator sequencing because DNA fragments of varying lengths are synthesized by incorporating both nucleotides and dideoxyterminators (deoxyribonucleotide triphosphates
[dNTPs] and dideoxynucleotide triphosphates [ddNTPs], respectively).
Amplification of the DNA was performed in a 50 [micro]l reaction composed of 0.2 mM deoxyribonucleotide triphosphates
(dNTPs) mix, 1.5 mM Mg[Cl.sub.2], 1U of Taq DNA polymerase (Fermentas), 10 pmol of each primer, and 100 ng of DNA from culture isolates.
The 25-[micro]L reaction mixture contained 0.1 [micro]M each of the primers, 50 ng of genomic DNA, 2.0 mM Mg[Cl.sub.2] 0.2 mM each of the deoxyribonucleotide triphosphates
, and 0.5 U of FastStart DNA polymerase (Roche Applied Science) in 1 x PCR buffer [50 mM Tris-HCl, 10 mM KCl, 50 mM [(NH4).sub.2]S[O.sub.4], pH 8.3 at 25[degrees]C].
The flu-2 COP-PCR mixture contained the same 5x buffer, 2 mM Mg[Cl.sub.2], the four deoxyribonucleotide triphosphates
, AmpliTaq DNA polymerase, and the flu-2 primers (same amounts as indicated for flu-1).