The combination of blocked-cleavable rhPCR primers, an enhanced Taq polymerase
, and a universal reporter system significantly increase specificity and boost signal, enabling the method to very accurately discriminate among extremely similar sequences.
Briefly, 5 u l of undiluted first RT-PCR product was added to a reaction mixture consisting of 3 u l of 25mM MgCl2, 2 u l of 10mM dNTPs, 0.5 u l of Taq Polymerase
(5U/ul), 5 u l of 10X Taq buffer, 1 u l of each type specific primers P, P and P and an upstream primer Con2 and RNase-free water to final volume of 50 l, and subjected to nested multiplex PCR amplification by following the PCR cycles as reported by Falcone et al.
As an early workhorse in PCR technology, Taq polymerase
has been studied extensively for purposes of fidelity determination.
Amplification reactions were performed in a volume of 25 [micro]L containing 1 [micro]L cDNA as template DNA, 0.5 [micro]M of each primer, 0.5 U Taq polymerase
(MBI, Fermentas), 0.1 [micro]M of each dNTP, and 1xx buffer for Taq polymerase
The PAL gene fragment generated by Taq polymerase
with sticky ends provided with 5'--A overhang at the insertion site which is more efficient.
For RAPD parameters like concentration of DNA template, primer, Magnesium chloride, Taq polymerase
, d NTP's and annealing Temperature were optimized which had an effect on amplification, banding pattern and reproducibility.
Amplification was conducted in 50-[micro]L volumes that contained 5 [micro]L of DNA, 30 [micro]L of distilled water, 10 [micro]L of 5x Taq buffer (Roche, Mannheim, Germany), 3 [micro]L of 25 mmol/L Mg[Cl.sub.2] (Roche), 1 [micro]L of 10 mmol/L deoxynucleotide triphosphates (Roche), 0.25 [micro]L of each primer (100 [micro]M), and 0.5 [micro]L (5 U/mL) of Taq polymerase
Each PCR reaction contained 0.5 M of each primer, 10-100 ng of extracted DNA, 1X Taq buffer, 0.8 mM dNTP, 2.5 mM magnesium, nad 0.05 units/l of Taq polymerase
. This mix was thermocycled for 30 cycles.
ReaX Assay 16S beads contain all the reagents required to perform 16S rRNAgene amplification, including a high-performance Taq polymerase
, dNTPs and the universal 16S rRNA gene-specific primers 27F and 907R.
Primary reactions contained 0.2 mM each dNTP, reaction buffer with Mg[Cl.sub.2] at 3 mM, 0.5 pmoles/mL each of primers HO1, HO3, Chlam5 (5'-CATTATGTCGGAGTCTGAGC), and Chlam3 (5'-GGATGACTCAAGGAATAGTCG), 1 mL of Internal Amplification Control (IAC) template (a DNA amplicon randomly amplified from the genome of a chicken with primer IAC whose concentration was undetermined), 0.4 pmoles/mL primer IAC (5'-TGTTTGACAGCTTATCAT), 5 mL DNA template preparation, 0.5 units of Taq polymerase
, and water to 25 mL.
Reviewing the literature on ancient bones, we found that contaminating humic acid and fulvic acid, both of which are found in wet soil, can inhibit the Taq polymerase
, the crucial factor in the PCR (Tsai et al.
Trizol RNA isolation reagent was obtained from Invitrogen (Karlsruhe, Germany), reverse transcriptase (murine lymphoma virus RT RNAseH-), random hexamer primers and Taq polymerase
, expressed in E.