High pure pcr product purification kit

Total DNA was extracted in triplicate from each cecal sample using a modified cetyltrimethylammonium bromide (CTAB) extraction method as previously described^{6 (link)}. This method uses the ionic detergent CTAB to disrupt cell membranes and a chloroform-isoamyl alcohol mixture that separates contaminants into the organic phase and nucleic acids into the aqueous phase. Resulting DNA was purified using a High Pure PCR product purification kit (Roche, Basel, Switzerland) according to the manufacturer’s instructions and was eluted in a final volume of 50 μL.
The V4-V6 region from the bacterial 16S rRNA operon was amplified from cecal DNA using a universal primer set, 16S-0515F (5′-TGYCAGCMGCCGCGGTA-3′) and 16S-1061R (5′-TCACGRCACGAGCTGACG-3′) tailed on each end with the Roche multiplex identifiers (MIDs). This barcode-based primer approach allowed sequencing of multiple samples in a single sequencing run without the need for physical partitioning. PCR conditions and reagents were similar to those described previously, and a standard concentration of 50 ng of cecal DNA was used in each reaction^{7 (link)}. PCR products were purified using a High Pure PCR product purification kit (Roche, Basel, Switzerland).

Since we retrieved only gene fragments of CD22 and MAG from our transcriptome of maraena whitefish, we derived primers from the 5′ and 3′ ends of the respective open reading frames. First, a SuperScript II Reverse Transcriptase Kit (Invitrogen/Thermo Fisher Scientific) was used to transcribe a total of 1 µg of RNA into cDNA. This reverse transcription was carried out at 42 °C for 50 min, followed by an inactivation step at 70 °C for 15 min. Purification of the cDNA was performed using a High Pure PCR Product Purification Kit (Roche), and the resulting cDNA was diluted in 100 µL of distilled water. Subsequently, we used the HotStarTaq Plus DNA Polymerase (Qiagen) to generate the PCR products of the full-length open reading frames. The purified (High Pure PCR Product Purification Kit; Roche) amplicons were inserted into a pGEM-T-Easy vector (Promega, Walldorf, Germany). The obtained plasmids were sequenced using the universal SP6/T7 primers and a MegaBACE capillary sequencer (GE Healthcare, Freiburg im Breisgau, Germany). Twelve clones were picked and analyzed per amplified sequence fragment.

DNA from collected clones was extracted using the Roche High Pure PCR Product Purification Kit (Roche, Pleasanton, CA, USA), and PCR was performed as previously described (Somboonna et al., 2008 (link)). All primers are listed in Supplementary Table S1. Clones were considered putative recombinants if they had the following characteristics by PCR: positive for the correct size band for the tet(C) gene; positive for the intergenic region (IGR) between the polymorphic membrane protein gene (pmp)B and pmpC using primers specific for S45; negative for the pmpC region using primers specific for Rogers132; and positive for the major outer membrane protein A gene (ompA) with confirmation of the S45 ompA genotype by Sanger sequencing.
Putative clonal recombinants were then propagated in 0.063 μg/mL tetracycline (4x MIC of S45) to grow stocks for whole-genome sequencing, MIC determination, and the tet(C) stability assay (see below). After the first passage, the harvest assay was performed a second time to ensure clonal purity. Picked clones were either grown as described above or directly picked and inoculated into 100 μL HBSS (Gibco) prior to DNA extraction, PCR, and ompA sequencing.