C. neoformans strains generated in this study are listed in S1 Table and were grown on YPD agar unless stated otherwise. All strains were created in C. neoformans type strain H99 [36 (link)]. Plasmid maintenance and cloning was carried out using the E. coli strain Mach1 (Life Technologies, USA). Plasmids generated in this study are listed in S2 Table .
Mach1
Manufactured by Thermo Fisher Scientific
Sourced in United States
The Mach1 is a high-performance microplate handler designed for automated sample processing in life science laboratories. It features a compact footprint and can accommodate a variety of microplate formats. The Mach1 enables efficient and reliable plate handling, ensuring consistent and reproducible results.
Automatically generated - may contain errors
Lab products found in correlation
Nuclease free water, by Qiagen (4 mentions)
Milli q purification system, by Merck Group (4 mentions)
Illustra templiphi 100 amplification kit, by GE Healthcare (4 mentions)
Phusion u green multiplex pcr master mix, by Thermo Fisher Scientific (3 mentions)
Qiaprep spin miniprep kit, by Qiagen (3 mentions)
Stellar, by Takara Bio (2 mentions)
Glucose, by MP Biomedicals (2 mentions)
Adenine hemisulfate, by MP Biomedicals (2 mentions)
Complete supplement mixture without tryptophan, by MP Biomedicals (2 mentions)
Eby100, by Thermo Fisher Scientific (2 mentions)
C thermocellum dsm1237, by American Type Culture Collection (2 mentions)
C cellulovorans, by American Type Culture Collection (2 mentions)
C cellulolyticum, by American Type Culture Collection (2 mentions)
R flavefaciens, by American Type Culture Collection (2 mentions)
Ammonium sulfate, by BD (2 mentions)
Recombinant eby100, by BD (2 mentions)
Superscript 3 first strand synthesis, by Thermo Fisher Scientific (2 mentions)
4d nucleofecor, by Lonza (2 mentions)
Neb turbo, by New England Biolabs (2 mentions)
Plasmid preparation kits, by Qiagen (2 mentions)
29 protocols using mach1
1
Cryptococcus neoformans Strain Generation
2
Bisulfite Sequencing of UR1A, UR1B, and UR6
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Bisulfite sequencing was used to analyze the following regions: K237 to C61 for UR1A (299 bp), K321 to K106 for UR1B (216 bp), and K2125 to K1787 (339 bp) for UR6. PCR was performed with the bisulfited DNA using primers listed in Table 4. Nested PCR amplified the regions UR1A and UR1B. The PCR products were cloned into pCR4-TOPO (Life Technologies), which was used to transform DH5a or TOP10 competent cells for bisulfited DNA from INS1 cells, or Mach1 (Life Technologies) for the linearized Beta-actin 4352931E R. norvegicus plasmid vector, followed by plating. Single colonies were selected, and the bisulfited DNA cloned into pCR4-TOPO was extracted using a NucleoSpin Plasmid QuickPure Kit (Macherey Nagel), followed by sequencing using the universal sequencing primer RVprimer3 or M13 reverse (5 0 -CAGGAAACAGCTATGAC-3 0 ). The data were analyzed using quantification tool for methylation analysis (QUMA; Kumaki et al. 2008) (link). The data are presented as the meanGS.E.M., and statistical significance was determined using a two-tailed unpaired Student's t-test. P!0.05 was considered to be significant. 1A). Interestingly, MafB mRNA was significantly increased, in these late-passage INS1 cells with p185 and p239 (Fig. 1B), similar to compromised b-cells in diabetic models, but not in INS1 cells with p42.
3
Cloning Plasmid DNA in E. coli
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MACH1 (Invitrogen) E. coli strain was used for passage during cloning of plasmid DNA.
4
Escherichia coli W Derivative Mach1 Protocol
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As a test organism we used the nonpathogenic Escherichia coli W (ATCC9637) derivative Mach1™ (T1 Phage-resistant, chemical competent, purchased form Invitrogen, USA) [43 ].
5
Fungal Strain Cultivation and Induction
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Fungal strains used in this study are listed in S21 Table . A. nidulans AGB551, which has a WT veA allele, was used for all deletion and epitope taggings. Stellar (Clontech) and MACH-1 (Invitrogen) competent Escherichia coli cells were used for recombinant DNA preparations. The WT and transformed A. nidulans strains were grown in Glucose Minimal Medium (GMM), supplemented with appropriate amounts of vitamins. For vegetative stage experiments, fungal spores were grown submerged in liquid GMM with 180 rpm rotation for 20 or 48h. For induction of development, vegetative mycelia were then filtered through miracloth and placed on solid GMM with 2% Agar. To induce the cultures asexually, cultures were grown vegetatively for 20h and shifted to the plates and were further incubated in the presence of light for 6, 12, 24h. For sexual induction, plates were covered by aluminium foil and incubated in the dark for 6, 12, 24, 48h. E. coli were grown in LB broth agar or liquid LB with Ampicillin (100 μg/ml) at 37°C overnight.
6
Yeast and Bacterial Strains for Protein Expression
7
Plasmid Construction Using USER and KLD
8
Cloning Plasmid DNA in E. coli
9
Propagation and Bioassay of Cucumber Mosaic Virus
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CMV-G2, a Hellenic isolate of CMV, obtained from a tomato plant (Solanum lycopersicum L.) exhibiting extreme leaf malformation and mottle, which was collected during a serious CMV disease outbreak in the area of Gastouni-Olympia (Greece) in 1998 [20 (link)], was used in this study. The isolate was preserved as dried tobacco leaf samples in CaCl2 and propagated in tobacco (Nicotiana tabacum) cv. Xanthi when needed.
The bacterial strain used for the in vivo production of dsRNA molecules was the E. coli strain HT115 (DE3), which has the RNase III gene disrupted by a Tn10 transposon carrying a tetracycline-resistance marker, as well as an IPTG-inducible T7 RNA polymerase gene contained within a stable insertion of a modified lambda prophage λ DE3 [68 (link)]. For all other molecular biology manipulations, the E. coli strains Mach1 (Invitrogen, Carlsbad, CA, USA) or DH5α were used.
The bioassays were performed on N tabacum cv. ‘Xanthi’ and Chenopodium quinoa plants. Seeds were sown and young plantlets at the stage of two leaves were transplanted in 8-cm-diameter pots. Plants were placed in a growth chamber with constant environmental conditions (temperature/photoperiod: 24 °C for 14 h of light and 18 °C for 10 h dark).
The bacterial strain used for the in vivo production of dsRNA molecules was the E. coli strain HT115 (DE3), which has the RNase III gene disrupted by a Tn10 transposon carrying a tetracycline-resistance marker, as well as an IPTG-inducible T7 RNA polymerase gene contained within a stable insertion of a modified lambda prophage λ DE3 [68 (link)]. For all other molecular biology manipulations, the E. coli strains Mach1 (Invitrogen, Carlsbad, CA, USA) or DH5α were used.
The bioassays were performed on N tabacum cv. ‘Xanthi’ and Chenopodium quinoa plants. Seeds were sown and young plantlets at the stage of two leaves were transplanted in 8-cm-diameter pots. Plants were placed in a growth chamber with constant environmental conditions (temperature/photoperiod: 24 °C for 14 h of light and 18 °C for 10 h dark).
10
Yeast and Bacterial Strains for Protein Expression
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The yeast S. cerevisiae strain EBY100 (Invitrogen, Carlsbad, CA) was used for mSEA surface display. The E. coli strains (Invitrogen) Mach1 and BL21 (DE3) were used for recombinant DNA manipulation and protein expression, respectively. All recombinant yeast and E. coli strains are summarized in Supplementary Table 1 . C. thermocellum DSM1237, C. cellulovorans, C. cellulolyticum and R. flavefaciens were purchased from ATCC (Manassas, VA) and cultured anaerobically following ATCC protocols. Recombinant EBY100 cells were cultured using SC-Trp medium: 1.67 g/L yeast nitrogen base without amino acids, 5 g/L ammonium sulfate (Difco Laboratories, Detroit, MI), 20 g/L glucose, 15 g/L adenine hemisulfate, and 0.64 g/L complete supplement mixture without tryptophan (MP Biomedicals, Solon, OH). Induction of aScaf display on yeast surface was performed in YPG media (1% yeast extract, 2% peptone, 2% galactose). E. coli was cultured in Luria-Bertani (LB) medium containing 50 μg/mL kanamycin. Unless otherwise indicated, all chemicals were purchased from Sigma-Aldrich (St. Louis, MO).
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