Positive samples were then subject to conventional PCR (cPCR). One analysis targeted the 16S rDNA gene (as described elsewhere: [35 (link)] and used a DreamTaq Green PCR Master Mix (2×) Kit (Thermo Fisher Scientific) in accordance with the manufacturer's instructions. A second analysis targeted the lfb1 gene (as described elsewhere: [36 (link)] and used a HotStarTaq DNA Polymerase Kit (Qiagen, Courtaboeuf, France) in accordance with the manufacturer's instructions. The amplified products were visualized using electrophoresis (1% agarose gel). Then, samples yielding clearly visible, high-intensity bands underwent Sanger sequencing, which was carried out by a service provider (Genoscreen, Lille, France). The Leptospira species present were identified through a Nucleotide BLAST search (http://blast.ncbi.nlm.nih.gov ).
Dreamtaq green pcr master mix 2 kit
Manufactured by Thermo Fisher Scientific
Sourced in United States
The DreamTaq Green PCR Master Mix (2×) Kit is a ready-to-use solution for performing polymerase chain reaction (PCR) amplification. The kit contains a specially formulated 2× master mix that includes the DreamTaq DNA Polymerase, dNTPs, MgCl2, and a green dye for direct gel loading.
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4 protocols using dreamtaq green pcr master mix 2 kit
1
Identification of Leptospira Species via PCR
2
Optimized DreamTaq Green PCR Protocol
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The DreamTaq Green PCR Master Mix (2×) kit, code number K1082 (Thermo ScientificTM), was utilized. 12.5 µl of DreamTaq Green PCR Master Mix, 7.5 µl of PCR-grade water, 1 µl of each forward and reverse 20 pmol primer, and 3 µl of template DNA make up the total amount of 25 µl/reaction.
3
D-loop Mitochondrial DNA Sequencing
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The primers used in this study were 5′-CCCCATGCTTACAAGCAAGT-3′ (nucleotides 16190–16209) and reverse 5′-GCTTTGAGGAGGTAAGCTAC-3′ (nucleotides 602–583) for amplification of a 982 bp product in the D-loop. DreamTaq Green PCR Master Mix Kit (2×) (Thermo Fisher Scientific, Waltham, MA, United States) was used for PCR and purification before sequencing. The BigDye Terminator v3.1 Cycle Sequencing Kit (Life Technologies, Carlsbad, CA, United States) was used to carry out cycle sequencing and the ABI PRISM Genetic Analyzer 3100 (Applied Biosystems) was used to separate the products. Polymorphisms were confirmed by repeated analysis of both strands. The Second Hospital of Hebei Medical University’s Human Tissue Research Committee approved all procedures.
4
Mitochondrial D-loop Amplification and Sequencing
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The primers used in this study were 5'-CCCCATGCTTACAAGCAAGT-3' (nucleotides 16,190–16,209) and reverse 5'-GCTTTGAGGAGGTAAGCTAC-3' (nucleotides 602–583), for the amplification of a 982 bp product in the D-loop. PCR was performed using the DreamTaq Green PCR Master Mix Kit (2 ×) (Thermo Fisher Scientific, Waltham, MA, USA) and purified before sequencing. Cycle sequencing was performed using the BigDye Terminator v3.1 Cycle Sequencing Kit (Life Technologies, Carlsbad, CA, USA), and the products were separated on an ABI PRISM Genetic Analyzer 3100 (Applied Biosystems, Foster City, CA, USA). Polymorphisms were confirmed by repeated analyses of both the strands.
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