To identify putative ΔEAS1 knockoffs the fungal transformants were screened by PCR as follows. DNA was extracted with the DNeasy 96 Plant Kit (Qiagen, Valencia, CA) and screened by PCR with primers specific for dmaW1 [dmaW1(f) and dmaWe19(-)-10] and dmaW2 (dmaWe19copy2.1d and dmaWe19copy2.5u). All of the putative knockoffs were also screened for the presence or absence of hph by PCR with the primer pair hph.3d and hph.3u. For complementation of the ΔEAS1 knockoff strain the transformants were screened for integration of the lpsB-containing plasmid by PCR with the primer pair 215hphlpsB(f) and 215lpsBhph(r). The PCR reactions were carried out in 25 μl reaction mixtures with 5–10 ng DNA template, 200 μM each dNTP, 0.2 μM each primer, 2.5 units AmpliTaq Gold, and AmpliTaq Gold PCR buffer with MgCl2 (1.5 mM final conc.) provided by the manufacturer (Applied Biosystems, Foster City, CA), in a model 2720 Thermal Cycler (Applied Biosystems). The temperature regime was as follows: 9 min at 95°, 35 cycles of 94° for 30 sec, annealing temperature (61° for dmaW2, 57° for lpsB-hph, 59° for dmaW1 and hph) for 35 sec, 72° for 2 min, and then a final 7 min incubation at 72°.
Amplitaq gold pcr buffer
Manufactured by Thermo Fisher Scientific
AmpliTaq Gold PCR buffer is a concentrated buffer solution designed for use in polymerase chain reaction (PCR) amplification. It provides the necessary components to facilitate the enzymatic amplification of DNA sequences.
Automatically generated - may contain errors
Lab products found in correlation
Amplitaq gold, by Thermo Fisher Scientific (3 mentions)
Dneasy 96 plant kit, by Qiagen (2 mentions)
Amplitaq gold dna polymerase, by Thermo Fisher Scientific (2 mentions)
Phusion hf buffer, by Thermo Fisher Scientific (1 mentions)
Phusion hot start high fidelity dna polymerase, by Thermo Fisher Scientific (1 mentions)
Geno grinder 2000, by SPEX (1 mentions)
Oligonucleotide, by Integrated DNA Technologies (1 mentions)
Zr fungal bacterial dna miniprep kit, by Zymo Research (1 mentions)
Rneasy plant mini kit, by Qiagen (1 mentions)
Zr plasmid miniprep classic kit, by Zymo Research (1 mentions)
Mgcl2, by Thermo Fisher Scientific (1 mentions)
Deoxynucleoside triphosphates dntps, by Thermo Fisher Scientific (1 mentions)
Molecular imager gel doc xr system, by Bio-Rad (1 mentions)
Nanodrop spectrophotometer, by Thermo Fisher Scientific (1 mentions)
Nucleospin 96 tissue kit, by Macherey-Nagel (1 mentions)
5 protocols using amplitaq gold pcr buffer
1
Screening Fungal Transformants for ΔEAS1 Knockoffs
2
Nucleic Acid Extraction and PCR Amplification
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Fungal DNA was isolated from fresh mycelium using ZR Fungal/Bacterial DNA MiniPrep kit (Zymo Research, Irvine, CA), or using Geno/Grinder 2000 (SPEX CertiPrep, Metuchen, NJ) and DNeasy 96 Plant Kit (Qiagen, Valencia, CA). Plasmid DNA was isolated from bacterial cultures using the ZR Plasmid Miniprep-Classic kit (Zymo Research, Irvine, CA). The mRNA was isolated from plant material using RNeasy Plant Mini Kit (Qiagen). PCR screens were performed using AmpliTaq Gold, and AmpliTaq Gold PCR buffer provided by the manufacturer (Applied Biosystems, Foster City, CA). For vector construction, the PCR amplifications were performed with Phusion Hot Start High-Fidelity DNA Polymerase (Thermo Scientific, Ratastie, Vantaa, Finland) with Phusion HF buffer (with 1.5 mM MgCl2) from the manufacturer. The temperature conditions were 98° for 3 min, followed by 35 cycles of 98° for 10 sec, 62° for 10 sec, and 72° for 7 min, then a final 5 min incubation at 72°. The oligonucleotides used in this study were from Integrated DNA Technologies (Coralville, IA) and are listed in Table 1 .
3
Error-Prone PCR for Tet Determinant Libraries
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In order to generate the libraries of mutagenized tet(A), tet(K), tet(M), and tet(X) sequences, 10 separate error-prone PCRs per gene were run with AmpliTaq Gold DNA polymerase (Applied Biosystems) in mutagenic buffer (29 (link)). In short, a 50-μl PCR mixture contained approximately 10 ng of linearized plasmid DNA, 1× AmpliTaq Gold PCR buffer (Applied Biosystems), 1.5 mM MgCl2 (Applied Biosystems), 0.2 mM deoxynucleoside triphosphates (dNTPs; Thermo Scientific), 0.3 μM forward primer and 0.3 µM reverse primer (see Table S1 in the supplemental material), and 5 U of DNA polymerase. In addition, the PCR mixture was supplemented with 4 or 6 μl of mutagenic buffer (4 mM dCTP [Thermo Scientific], 4 mM dTTP [Thermo Scientific], 27.5 mM MgCl2 [Thermo Scientific], and 2.5 mM MnCl2) to vary the mutation rate. The PCR mixture was subjected to 2 min of initial denaturation at 94°C followed by 25 cycles of 1 min of denaturation at 94°C and 2 min of annealing and elongation at 72°C. A final elongation step was performed for 10 min at 72°C. After confirmation of PCR products with agarose gel electrophoresis, 10 reaction products were pooled to form the tet sequence library. Two sequence libraries (using 4 or 6 μl of mutagenic buffer) were generated per Tet determinant.
4
Molecular Characterization of ST258 Isolates
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A total of 9 primer pairs were designed based upon K-41 and K-28 WGS assemblies to confirm and determine gene content among ST258 isolates (S2 Table ). Six primer pairs specific to genes flanking or within a 45-kb contiguous region in K-41 were used to demonstrate gene presence or absence by PCR (S1 Fig ). Two additional primer sets were used to determine the presence or absence of trehalose synthase or nitrate sensor/regulator genes among ST258 isolates by PCR. Finally, primers specific to a newly described KPC-encoding plasmid, pKp28, were designed to determine plasmid presence or absence among ST258 isolates by PCR. All PCR reactions were performed in 50 ul with 1X AmpliTaq Gold PCR buffer, 2.5mM MgCl2, 0.2mM each deoxynucleoside triphosphate, 0.2 uM each primer and 1.5 U AmpliTaq Gold DNA polymerase (Applied Biosystems, Foster City, CA). PCR cycle conditions were: 95°C for 5 min., followed by 35 cycles of 95°C for 1 min, 54°C for 1 min, 72°C for 1.5 min and a final extension at 72°C for 7 min.
5
Extraction and Detection of Hematodinium perezi DNA
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DNA initially was extracted from ~2 mg of freezedried muscle tissue, ~200 µl of preserved hemolymph, or from whole ethanol-preserved megalopae using NucleoSpin ® 96 Tissue Kits (Machery-Nagel) with an epMotion 5075 TMX liquid handling workstation (Eppendorf) following the manufacturer's protocol. The presence of DNA of H. perezi was detected by PCR amplification of a portion of the 18s ribosomal RNA gene using the primers Hemat-F-18S and Hemat-R-18S developed by Friedman et al. (2009) . For samples in which DNA of H. perezi was detected, DNA was subsequently extracted from hepatopancreas, ovary and, where available, egg-mass tissue using the same protocol. DNA concentrations were determined with a Nanodrop spectrophotometer (Thermo Scientific). PCR reactions were in 15 µl with 1× AmpliTaq Gold ® PCR Buffer (Applied Biosystems), 2.5 mM MgCl 2 , (1 mM) dNTPs, (1.2 µM) of each forward and reverse primer, 0.6 units of AmpliTaq ® Gold (Applied Biosystems), 20 ng of DNA, and Milli-Q ® water. PCR conditions were as follows: 95°C for 5 min, then 40 cycles of 96°C for 15 s, 56°C for 30 s, 72°C for 45 s, and lastly 72°C for 10 min. Positive and negative controls were included in all reactions. PCR products were electrophoresed on 2% agarose gels with 0.05% ethidium bromide and visualized on a Molecular Imager ® Gel Doc ™ XR system (Bio Rad).
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