Ultrapure salmon sperm dna solution

If not otherwise stated, chemical reagents were purchased from Sigma-Aldrich (Vienna, Austria). Denatured ethanol (EtOH) was purchased from Carl Roth (Vienna, Austria), deionized formamide was purchased from PanReac AppliChem (Darmstadt, Germany), EtOH absolute was purchased from AustrAlco (Spillern, Austria), and UltraPure salmon sperm DNA solution was purchased from Thermo Fisher Scientific (Vienna, Austria). Cell culture media, media supplements and antibiotics were purchased from Thermo Fisher Scientific. All buffers were prepared using ultrapure water (Milli-Q, 18.2 MΩ·cm at 25 °C, Merck Millipore, Vienna, Austria) and filtered through sterile 0.22 µm polyvinylidene fluoride (PVDF) syringe filters (Carl Roth, Austria). Surfaces and instruments were wiped with the surface decontaminant RNase Away (Carl Roth, Austria) prior to work.

Francisella capsule was isolated by subjecting EtOHp to a modified hot phenol/water extraction followed by water/Triton X-114 partitioning and SDS gel filtration as described previously [4 (link)] (S1A Fig). An alternate protocol to isolate capsular polysaccharides from the nuclease-treated EtOHp (S1B Fig) was developed using deoxycholate (DOC)-based gel sieving [12 (link)]. EtOHp samples were dissolved in 2% DOC in 0.2 M NaCl, 50 mM Tris, 5 mM EDTA and sonicated for 30 min in a bath sonicator. After centrifugation to remove any insoluble debris, the sample was applied to a 16 mm x 30 cm Sephacryl S-200 column on an Akta Purifier FPLC system (GE Healthcare) at a flow rate of 0.5 ml/min. In order to remove any residual DOC that might interfere with subsequent chromatographic analysis, fractions underwent three rounds of ethanol precipitation as described above after addition of purified DNA (150 μg/ml UltraPure™ Salmon Sperm DNA Solution, Invitrogen) to enhance recovery. To control for any effects of the DOC or ethanol washing on subsequent chromatographic analysis, whole EtOHp was also treated in the same way in parallel with isolated capsule or low molecular weight material. Washed samples were raised in water and stored at 4°C until further analysis.

After sampling, the harvested leaves were air dried. Approximately 2–4 mg dried leaf material from 2–3 cm above and below the region where infective gauze bandage had been placed was first ground in a ball mill (Retsch, Germany) at maximal speed (4×8 min.). DNA extractions of L. zosterae were performed with an Invisorb spin tissue mini kit (Invitek, Berlin, Germany) following the manufacturer's instructions. To enhance extraction efficiency and to ensure that even low amounts of target DNA were carried through the filter absorption steps, 1 µL (containing ∼500 ng) of UltraPure salmon sperm DNA solution (Invitrogen, Life Technologies, USA) was added to each extraction to saturate silica columns with DNA. Target DNA was purified using a one-step PCR inhibitor removal kit (Zymo Research, USA).
To determine Labyrinthula zosterae cell number, we followed a TaqMan based rt-QPCR assay as described in Bockelmann et al.[18] (link) with a fluorescently-labeled ITS probe.
In one reaction we used 10 µL TaqMan universal Master Mix (Applied Biosystems, now Life Technologies) in a 20 µL reaction volume: 2 µL 1∶10 diluted template DNA, 2.4 µL (40.8 nM) of the two primers, 2.4 µL Milli-Q H₂O and 0.8 µL probe (50 nM), respectively. The thermo-cycling program on a Step-One QPCR machine was 2 min at 50°C and 10 min at 95°C, followed by 48 cycles at 95°C for 15 s and 1 min at 60°C.

Genes frequently deleted in the general population were selected for the study (see Table 1 for frequency of null genotype): SRY, GSTM1, GSTT1, RHD, LEC3C, and TRY6, and their expression evaluated by qPCR in both the donor cells and the LCT recipients.
To this end, FAM-labelled assays for the gene of interest were used in duplex with the HEX-labelled assay for the reference gene RNAse P (Integrated DNA Technologies (IDT), Leuven, Belgium). Sequences of the primers and probes used can be found in Table 2.
PCR was performed in a total volume of 20 μl containing TaqMan® Gene Expression Master Mix (Applied Biosystems, Thermo Fisher Scientific), primers and probes for the gene of interest and the reference gene, and 30-120 ng of genomic DNA and Tris-EDTA (TE) buffer (IDT).
The thermocycling was performed with adequate controls on a LightCycler® 480 System (Roche Diagnostics, Basel, Switzerland) with the following protocol: incubation for 2 min at 50°C, followed by denaturation for 10 min at 95°C and 40 amplification cycles of 15 s at 95°C and 1 min at 60°C (acquisition). gBlocks gene fragments were used as positive controls (IDT). An ultrapure salmon sperm DNA solution (Invitrogen, Thermo Fisher Scientific) was used as a negative control, as well as “no template controls,” wherein the DNA was replaced by TE buffer.

Vectors were linearized by restriction digestion, and purified and concentrated by ethanol precipitation. N. oceanica CCMP1779 transformation was performed according to the method of Vieler et al. (2012) with 3 μg of vector DNA, with 30 μg carrier DNA (Invitrogen UltraPure™ Salmon Sperm DNA Solution). Transformed cells were allowed to recover for 48 h and then plated in top agar with the respective selection. After 3–4 weeks, individual colonies were resuspended in 100 μL F/2. From each transformation, ~20 colonies were screened for increased LC‐PUFA content, and two to three colonies identified as positive.

Dextran sulfate sodium (MW ca 40,000 Da, cat no. J63606) was purchased from Alfa Aesar. RIPA Lysis Buffer (cat no. 89900) and Halt™ Protease Inhibitor Cocktail (cat no. 87786) were purchased from Thermo Scientific™. Dopamine hydrochloride (cat no. H8502) and Phorbol 12-myristate 13-acetate (PMA, cat no. 8139) were purchased from Sigma Aldrich. DNase-I (cat no. 10104159001), Collagenase D (cat no. COLLD-RO), and 1,4-dithiothreitol (DTT, cat no. DTT-RO) were purchased from Roche. Mesalamine (cat no. PHR1060) was purchased from Supleco. TRIzol™ Reagent (cat no. 15596026), RNAlater™ Stabilization Solution (cat no. AM7020), DNase I Buffer (cat no. AM8170G), Ambion™ DNase I (cat no. AM2222), and UltraPure™ Salmon Sperm DNA Solution (cat no. 15632011) were purchased from Invitrogen™. Percoll™ (Density 1.130 g/mL, cat no. 17089101) was purchased from Cytiva. Poly(lactic-co-glycolic acid) (50/50 ratio, inherent viscosity 0.55–0.75, acid terminated, cat no. B6013-2P) was purchased from LACTEL® Absorbable Polymers. 4arm polyethylene glycol (HCL salt, MW 5000, cat no. 4ARM-NH₂ 5000) was purchased from JenKem Technology.