Near full-length 16S rRNA genes were amplified in 27 PCR cycles with Taq Master Mix RED (Ampliqon A/S) and the general bacterial primer 8F with ambiguities, 616V according to Juretschko et al. (1998 (link)) (5′-AGA GTT TGA TYM TGG CTC AG-3′) and universal primer Uni1492R (5′-GGY TAC CTT GTT ACG ACT T-3′; Loy et al., 2002 (link)) at an annealing temperature of 52°C. PCR products were cloned into E. coli JM109 using the pGEM-T vector system (Promega), and 168 clones were selected (96 from the fresh 2011 sample and 24 clones from each of the ethanol fixed samples) for partial Sanger sequencing (GATC Biotech). A few clones representing the phylotype of the putative symbiont were selected from each clone library for almost full-length sequencing. The 16S rRNA gene sequences of the symbiont were deposited in GenBank under the accession numbers KP138714 , KP138715 , KP138716 (full-lengths), and KU195266 (900 bp).
Taq master mix red
Manufactured by Ampliqon
Sourced in Denmark
Taq Master Mix RED is a ready-to-use solution for PCR amplification. It contains Taq DNA polymerase, dNTPs, and all necessary components for PCR, except primers and template DNA.
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Lab products found in correlation
2 protocols using taq master mix red
1
Sequencing Bacterial 16S rRNA Gene
2
EGFR Gene Exons Amplification and Sequencing
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Exons 18, 19, 20, and 21 of the EGFR gene were amplified with minor modification; namely, nested polymerase chain reaction (PCR) was only performed on specimens when their first PCR products could not be visualized on 2% agarose gel electrophoresis. The first round of PCR was performed in a total volume of 25 μL containing 2 μL of DNA, 1× Taq Master Mix Red (Ampliqon III, Odense, Denmark), and 0.5 μM of each primer. This PCR program consisted of 35 cycles of (95°C for 40 s, 56°C for 40 s, and 72°C for 40 s), followed by a 5-minute extension stage at 72°C. For the nested PCR protocol, DNA amplification was performed using the same PCR program as described above, using 2 μL of the first PCR products as a template, 1× Taq Master Mix Red, and 0.5 μM of each primer. Sanger sequencing was performed with forward or reverse primers, and sequence analyses were performed using Mutation Surveyor software (SoftGenetics, State College, PA, USA).
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