The 50ul amplification reaction mixture consisted of 1x PCR buffer (75mM Tris-HCl pH 8.8, 20 mM (NH4)2SO4 and 0.01% tween 20), 0.125mM dNTPs mixture, 1.5mM magnesium chloride, 100 pmoles of each forward and reverse primer, 2.5 units of Taq DNA polymerase (Fermentas
Cat # EP0402) and 100ng of template (genomic DNA).
PCR experiments were conducted in a volume of 25 [MICRO]L, consisting of 1X PCR buffer (Fermentas
, GmbH, Germany), 2 mM Mg[Cl.sub.2], 0.2 mM dNTPs, 0.5 [micro]M of the non-biotinylated universal bacterial primers, 50 ng of genomic DNA (each of the five target bacteria) and 1 unit Taq DNA polymerase (Fermentas
, GmbH, Germany).
PCR reactions were prepared in 25 uL volume consisting of 1X Taq Buffer (Fermentas
), 2 uM MgCl2 (Fermentas
), 2.5 uM dNTP, 2.5 uM primer, 6 ng of genomic DNA, 1U of Taq Polymerase (Fermentas
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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.
The PCR reaction was performed using 2.5 [micro]L 10X buffer (MBI Fermentas
), 1 [micro]L of 2.5 mM dNTP (Invitrogen, USA), 1.5 [micro]L of 25 mM MgCE (MBI Fermentas
), 2 [micro]L of the 10 pmol EI1 primer (Invitrogen, USA), 0.2 [micro]L of 1.25 U taq DNA polymerase (MBI Fermentas
) and 1.0 [micro]L of DNA.
Both the insert and vector were ligated using T4-Ligase Rapid kit (Fermentas
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Hundred uL of reaction mixture was made up of 7 uL dNTPs, 5 uL of 10x PCR buffer, 3 uL MgCl2, 2 uL of each primer, 1.5 uLTaq polymerase (Fermentas
, USA), 10 uL template DNA and 10 mM Tris buffer (pH 8.3) to make the volume.
RNA extraction (Gene Jet RNA Purification Kit, Fermentas
) and complementary doxyribonucleic acid (cDNA synthesis (RevertAid Premium First Strand DNA, Fermentas
) was carried out as per manufacturer's protocol.
 PCR reaction was carried out in 20 [micro]L reaction volume, containing 50ng of template DNA, 1.5 mM Mg[Cl.sub.2], 0.2 mM dNTPs (Fermentas
, USA), 10 picomoles of each primer, 1U of Taq polymerase (Sigma-Aldrich, India) and 1X Taq buffer.
The DNA (stock concentration 100 [micro]mol/L) oligonucleotides were mixed to a final concentration of 10 [micro]mol/L in 1X [T.sub.4] polynucleotide kinase reaction buffer A (Fermentas
) with 0.25 U/[micro]L T4 polynucleotide kinase (Fermentas
), 10 mmol/L ATP, and incubated for 30 min at 37[degrees]C, followed by inactivation at 75[degrees]C for 10 min in a thermocycler.