PCR amplification of ITS2 of the rRNA gene and two mitochondrial genes, COI and 16S rRNA, was performed, using both specifically designed primers and previously published primers (Table-1 ) [18 (link)-20 (link)]. H. dromedarii DNA (2 μl) and 0.3 μl of each primer (10 pmol) (Macrogen, Seoul, South Korea) were added to the PCR mixture, containing one unit of Max Taq DNA Polymerase (Vivantis Technologies, Malaysia), 5 μl 10× ViBuffer (Vivantis Technologies, Malaysia), and 2 μl dNTPs (10 mM). The volume was adjusted to 25 μl by adding distilled water. Thermal cycling was performed on a TPersonal Thermocycler (BIOMETRA, Germany) with an initial 15 min cycle at 95°C, followed by 35 cycles consisting of 30 s at 94°C, 1 min at 55°C or 60°C depending on the primers, and 1 min at 72°C, followed by a final 10 min extension step at 72°C. To rule out DNA or amplicon contamination, Molecular Grade Water was used as a negative control throughout each step of the protocol. The PCR amplicons were sequenced by Macrogen Inc. (Seoul, South Korea) using BigDye (Applied Biosystems, California, USA) on an ABI3730XL DNA sequencer (Applied Biosystems, California, USA).
Max taq dna polymerase
Manufactured by Vivantis Technologies
Sourced in Malaysia
Max Taq DNA Polymerase is a thermostable DNA polymerase enzyme used for DNA amplification in polymerase chain reaction (PCR) techniques. It catalyzes the synthesis of new DNA strands complementary to a template DNA sequence.
Automatically generated - may contain errors
2 protocols using max taq dna polymerase
1
Molecular Identification of Dromedary Tick
2
Cloning of cry1Ab Gene from Bt SY49-1
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Total DNA of Bt SY49-1 was extracted according to the method of Bravo et al.8 (link) and used as template for polymerase chain reactions (PCR). The cry1Ab gene was amplified using the primer pairs F-5′-GGA TCC ATG GAT AAC AAT CCG AAC ATC-‘3; R- 5′-GTC GAC TTA TTC CTC CAT AAG AAG TAA-3′.9 (link)
BamHI and SalI restriction enzyme recognition sequences were added to the 5′ end of the forward and reverse primers, respectively. PCR mixes contained the reagents at a final concentration of 2.3 mM MgCl2, 1× Taq buffer, 0.2 mM dNTP mix, 0.3 pmol of each primer, 0.5 U MaxTaq DNA polymerase (Vivantis, PL2201), and 30–100 ng template DNA. The PCR amplification was performed under the following conditions: Initial denaturation at 95 °C for 5 min, followed by 30 cycles at 94 °C for 1 min, 52 °C for 1 min, 72 °C for 3.5 min, and a final extension step at 72 °C for 10 min.9 (link) Adenine was added to the 3′ end of the PCR products after amplification for the TA cloning process.
BamHI and SalI restriction enzyme recognition sequences were added to the 5′ end of the forward and reverse primers, respectively. PCR mixes contained the reagents at a final concentration of 2.3 mM MgCl2, 1× Taq buffer, 0.2 mM dNTP mix, 0.3 pmol of each primer, 0.5 U MaxTaq DNA polymerase (Vivantis, PL2201), and 30–100 ng template DNA. The PCR amplification was performed under the following conditions: Initial denaturation at 95 °C for 5 min, followed by 30 cycles at 94 °C for 1 min, 52 °C for 1 min, 72 °C for 3.5 min, and a final extension step at 72 °C for 10 min.9 (link) Adenine was added to the 3′ end of the PCR products after amplification for the TA cloning process.
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