Abi prism 3730xl dna

Genomic DNA extraction was carried out as described previously [64 (link)], from cultures grown on 50 mL glass flasks with 30 mL of BG-11 media under conditions mentioned above for 4 days. Genomic DNA was analyzed by Macrogen Inc (South Korea) for amplification of 16S rRNA gene using the primers 785F (5′-GGATTAGATACCCTGGTA-3′), 907R (5′-CCGTCAATTCMTTTRAGTTT-3′), 27F (5′-AGAGTTTGATCMTGGCTCAG-3′), and 1492R (5′-TACGGYTACCTTGTTACGACTT-3′). PCR products were sequenced by Sanger [65 (link)], using an ABI Prism 3730XL DNA analyzer (Applied Biosystems, Macrogen Inc, Seoul, South Korea). The sequences obtained were analyzed using Basic Local Alignment Search Tool (BLASTN) and matched against non-redundant nucleotide collection (nr/nt) present in NCBI GenBank database (http://www.ncbi.nlm.nih.gov).

Genomic DNA of newly sequenced Norway rats was prepared from liver tissue using the DNeasy tissue kit (Qiagen, Valencia, CA, USA). The cyt-b of rats from Germany/France was amplified using published primers [27] . D-loop sequences of rats from Germany were amplified using the forward and reverse primers (5′- TCA GGA CAA TCA AGA AGA AGG A -3′ and 5′- TGA GGG TAG GCA AGT AAA GAG G-3′; designed using Primer 3 [28] (link)). M13 sequencing tails (5′- CACGACGTTGTAAAACGAC-3′ and 5′- GGATAACAATTTCACACAGG-3′) were attached to the 5′-ends of forward and reverse primers. D-loop sequences of rats from France were amplified using published primers [27] .
PCR thermo-cycling conditions generally were as follows: 2 minutes at 94°C, followed by 30 cycles of 30 seconds at 94°C, 30 seconds at 56°C, 1 minute at 72°C, and a final 5 minutes at 72°C. Products were cleaned using ExoSAP-IT (USB, Cleveland, OH, USA), sequenced in both directions (Sanger method), and read on an ABI Prism™ 3730xl DNA (Applied Biosystems, Foster City, CA, USA). Reads were assembled, proofread and aligned to the corresponding regions of the mtDNA of R. norvegicus (GenBank No. NC_001665) using the software Lasergene 7.2 [29] (link). Missing bases of shorter sequences were treated following the minimal distance rule [30] (link).