Illustra puretaq ready to go pcr beads

A part of the bacterial suspension from relapsing mice was inactivated at 95°C during 30 min, to allow bacterial DNA extraction by heat shock (5 cycles of 2 min at 95°C and 2 min at 4°C) following by 15 min at 95°C and 15 min in an ultrasonic bath.
IllustraTM PuReTaq Ready-To-Go PCR beads (GE Healthcare) was used to perform PCRs. For the rpoB gene, we used primers as previously reported [17 (link)] RPOBMuS 5’-GCGCACGGTGGGTGAGCTG-3’ RPOBMuAS 5’-CGAGACGCCCTACCGCAAGG-3’. Other primers were designed according to information on the complete genome of M. ulcerans (GenBank accession number CP000325): for the gyrA gene, MugyrAS 5’-CGCCGTGTGCTCTATGCCATG-3’ and MugyrAAS TCGCCGGGTAATGACCCGCCA-3’, and for the ARN23S gene 23.1, 5’-AATGGCGTAACGACTTCTCAACTGT-3’ and 23.2 5’-GCACTAGAGGTTCGTCCGTCCC-3’.
PCR-amplified fragment were purified by using Qiagen DNA purification kit (Qiagen) and sequenced by the dideoxychain termination method with the ABI PRISM BigDye Terminator V3.1 Cycle Sequencing Kit (Life Technologies). The oligonulceotide primers used for DNA sequencing were the same as those used for PCR. The sequences were analyzed with the software Seqscape 2.0 (Life Technologies).

Total genomic DNA was isolated using DNeasy Tissue Kits (Qiagen, Valencia CA). We amplified and sequenced a 636 bp fragment of the mitochondrial cytochrome c oxidase subunit I (COI) gene using primers HCO2198 and LCO1490 [48 ] or primers VF1 and VR1 [49 ]. Bats from all sample sites except Michigan were sequenced, for a total of 617 individuals. PCRs were conducted in 25 μl volumes containing 0.4 μM of each primer and 20–50 ng of DNA template, using Illustra PuReTaq Ready-To-Go PCR beads (GE Healthcare Life Sciences, Pittsburgh PA). When reconstituted to 25 μl with water, these beads contained 2.5 units PuReTaq DNA polymerase, 200 μM each dNTP in 10 mM Tris-HCl (pH 9), 50 mM KCl, 1.5 mM MgCl₂, and an unspecified concentration of bovine serum albumin (BSA). No other additives were added to the solution. Cycling conditions consisted of one cycle of 5 min at 94°C, 30 cycles of 30 sec at 94°C, 45 sec at 68°C and 1 min at 72°C, and a final cycle of 2 min at 72°C. PCR products were purified by digestion with exonuclease I and shrimp alkaline phosphatase (EXOSAP), and were sequenced in both directions, using the amplification primers, at the University of Arizona Genetics Core Facility. Sequences were edited using CodonCode Aligner 3.0 (Gene Codes Corp.) and aligned using the default settings in MAFFT [50 (link)].

For MO validation by RT-PCR, total RNAs from control and injected embryos were extracted with RNeasy Micro Kit (QIAGEN) and reverse-transcribed using SuperScript IV VILO Master Mix (Thermo Fisher Scientific) according to the manufacturer’s instructions, and used for PCR with Illustra PuReTaq Ready-To-Go PCR beads (GE Healthcare, Chicago, IL). The following primer sets spanning the entire coding sequence were used:

Cells were plated on LA (5 g/L yeast extract, 10 g/L tryptone, 10 g/L NaCl, 15 g/L agar) with 0.2% glucose directly after the spacer acquisition assay and grown overnight at 37 °C. Fluorescent colonies were visualized in ChemiDoc^TM MP with Image lab v. 4.0 software (Bio-Rad) using Alexa 546 settings (excitation: blue epi illumination, emission: 605/50 filter). For selected colonies, the CRISPR array was investigated by colony PCR, using the same primers as above, and Illustra PuRe Taq Ready-to-Go PCR beads (GE Healthcare). The same colonies were re-streaked to confirm the fluorescent phenotype. 5 µl PCR product was analyzed on 1.5% agarose TBE gel with SYBR safe (Thermo Fisher Scientific) and visualized in ChemiDoc^TM MP (Bio-Rad) as described above.

A similarly-sized small piece of kidney and/or gill tissue was cut off and lysed overnight at 56 °C in 100 μl of Mole® lysis buffer and 10 μl of Proteinase K solution (Sigma-Aldrich, Hamburg, Germany). DNA from each piece was then extracted on a GenMole (Mole Genetics, Oslo, Norway) using the Mole Genetics DNA Tissue Kit and the DNA tissue protocol. Approximately 900 bp of rDNA was amplified using the primers and protocol of Freeman et al. [22 (link)] as described below. All PCR reactions were done using Illustra PuReTaq Ready-To-Go ™ PCR Beads (GE Healthcare, Oslo, Norway). Each reaction contained 1 μl of the forward primer, 1 μl of the reverse primer, 3 μl of the template DNA and 20 μl of sterile water. PCR products were sent to Macrogen for sequencing (Macrogen, Amsterdam, The Netherlands). All products were sequenced using the PCR primers. Sequences were proofread in VectorNTI ver. 11 (Invitrogen, Oslo, Norway) and subjected to a GenBank BLASTn search to search for identity with known sequences. The same samples were also subjected to RT-PCR analyses following the protocol of Nylund et al. [29 (link)], but targeting the rRNA gene and each analysis was repeated once. Average values for replicates were calculated.