An AZ100 Microscope (Nikon, Tokyo, Japan) with a GX1920 CCD camera (Allied Vision Technologies, Burnaby, BC, Canada) was used to acquire brightfield and fluorescence images of cells and droplets. A high-speed GC640 CCD camera (Allied Vision Technologies) was used to record the formation of double emulsion droplets and aqueous droplets. Multi-well plate screening experiments were performed using a Typhoon FLA 9000 imaging system (GE Healthcare, Chicago, IL). Images were analyzed using ImageJ (http://rsbweb.nih.gov ).Goldwave software (http://www.goldwave.com ) was used to analyze sound data collected during stage movement.
Typhoon fla 9000 imaging system
Manufactured by GE Healthcare
The Typhoon FLA 9000 imaging system is a high-performance fluorescence imager designed for a wide range of applications in life science research. It provides sensitive detection and quantification of fluorescent proteins, dyes, and other fluorescent molecules in various sample types such as gels, blots, and microplates.
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Lab products found in correlation
Dnase 1, by Thermo Fisher Scientific (2 mentions)
Az100 microscope, by Nikon (1 mentions)
Bovine serum albumin (bsa), by Thermo Fisher Scientific (1 mentions)
Cfx96 real time pcr instrument, by Bio-Rad (1 mentions)
Abil we09, by Evonik (1 mentions)
Tegosoft dec, by Evonik (1 mentions)
Realtime hiv 1 assay, by Abbott (1 mentions)
Bis tris acrylamide gel, by Thermo Fisher Scientific (1 mentions)
Nativepage running buffer, by Thermo Fisher Scientific (1 mentions)
Polyethyleneimine cellulose tlc plates, by Merck Group (1 mentions)
3mm filter paper, by Cytiva (1 mentions)
γ 32p atp, by PerkinElmer (1 mentions)
Acrylamide, by Bio-Rad (1 mentions)
Genosmart gel documentation system, by Avantor (1 mentions)
Hybond n membrane, by GE Healthcare (1 mentions)
Chef dr 3 system, by Bio-Rad (1 mentions)
8 protocols using typhoon fla 9000 imaging system
1
Multimodal Imaging of Microfluidic Droplets
2
Quantitative NASBA for HIV-1 RNA Detection
3
SLFN5 Interaction with Nucleic Acids
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EMSAs were conducted to analyze the interaction between SLFN5 and various nucleic acid substrates. Increasing amounts of SLFN5 were titrated to fluorescently labeled substrates (6-FAM or Cy5 labelled, 40 nM) in EMSA buffer (25 mM HEPES pH 7.0, 60 mM NaCl, 5 % glycerol, 2 mM MgCl2, 0.5 mM DTT) and incubated on ice for 30 minutes. The samples were analysed by native PAGE on 3–12 % acrylamide Bis-Tris gels (Invitrogen). The electrophoresis was performed in 1× NativePAGE Running Buffer (Invitrogen) at 4°C and 100 V for 120 min. The gels were visualized using a Typhoon FLA 9000 imaging system (GE healthcare). The images were analysed and integrated using GIMP v2.10.2 and ImageJ (72 (link)).
4
ATPase Assay of SLFN5 Protein
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For the ATPase assay 5 μM SLFN51–336 was incubated in presence or absence of 0.2 μM single-stranded 60-mer poly (dT) DNA in 50 mM Tris pH 7.5, 150 mM KCl, 5 mM MgCl2, 1.5 mM ATP and 10 nM [γ-32P] ATP (Hartmann Analytik, Germany) at 37°C for 0 or 60 min. For analysis, 1 μl of the reaction mixture was applied onto polyethyleneimine cellulose TLC plates (Sigma-Aldrich, Germany) and free phosphate was separated from ATP by thin layer chromatography in TLC running buffer (1 M formic acid, 0.5 M LiCl). [γ-32P] ATP was detected using a Typhoon FLA 9000 imaging system (GE healthcare).
5
Quantification of pre- and mature rRNA levels
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Steady-state levels of pre- and mature rRNAs were assessed by northern and primer extension analyses as described previously (48 (link),50 ). Specific oligonucleotides, whose sequences are provided in Supplementary Table S3 , were 5′-end labelled with 20 μCi of [γ-32P]ATP (6000 Ci/mmol; Perkin Elmer) and used as probes or primers. In all experiments, total RNA was extracted from samples corresponding to 10 OD600 units of exponentially grown cells. Equal amounts of total RNA (5 μg) were loaded and resolved on 1.2% agarose–6% formaldehyde and 7% polyacrylamide–8 M urea gels. Then, RNA was transferred to and immobilized on Hybond-N nylon membranes and subjected to hybridization (48 (link),50 ). Primer extension was done with the same RNA samples as those used for northern blot analysis according to Venema et al. (50 ). The resulting cDNA products were resolved on 6% polyacrylamide–8 M urea gels, which were then dried on Whatman 3MM filter paper. Both membranes and filter papers were subjected to phosphorimager analysis with a Typhoon™ FLA9000 imaging system (GE Healthcare).
6
Schlafen Protein Nuclease Activity Assay
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The 6-FAM labeled nucleic acid substrate (tRNASer or DNA) (50 nM) was incubated with the indicated Schlafen protein (250 nM) in nuclease buffer (25 mM Tris pH 7.3, 120 mM NaCl, 4 mM MgCl2, 1 mM DTT) at 37°C for 45 min. Where indicated, MnCl2 (2 mM) or EDTA (10 mM) was added. For the nuclease assay with DNA as a substrate, DNase I (Thermo Fisher Scientific) was used as positive control. The samples were analyzed on 15% denaturing polyacrylamide gels (Rotiphorese® DNA sequencing system) in 1× TBE buffer. Gels were run at 270 V for 50 min and visualized using a Typhoon FLA 9000 imaging system (GE healthcare). The images were analysed and integrated using GIMP v2.10.2.
7
Optimal Binding Conditions for Gel-Shift Assays
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Gels were prepared with a final concentration of 10% acrylamide (Bio-Rad) in the running gel and 5% in the stacking gel. The native running buffer contained 25 mM Tris-HCl, pH 8.3, and 192 mM glycine. The native loading buffer was made with 62.5 mM Tris-HCl, pH 6.8, 25% glycerol and 1% bromophenol blue dye. Samples for the gel-shift assay were prepared with 5 μg of protein per sample. For the VPS gradient, 0, 0.0625, 0.125, 0.25, 0.50, 1, and 5 µg of VPS was preincubated with the representative protein for 5 min. For the BSM gradient, 0, 0.5, 1, 2, 4, 6, 8, and 10 μg of BSM was preincubated with RbmCM1M2 for 5 min. For the GFP control, the highest amount of VPS or BSM was used. For testing different polysaccharides, 5 µg (highest amount used in the VPS concentration gradients) was preincubated with the Bap1Δ57aa (5 μg) for 5 min. Gel electrophoresis was performed at 85 V for 4 h in an ice bath. Images were acquired on gels (still encapsulated in glass) with an excitation wavelength of 492 nm and an emission wavelength of 513 nm using a Typhoon FLA 9000 imaging system (GE Healthcare).
8
Genomic Analysis via Pulsed-Field Gel Electrophoresis
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Pulsed-field gel electrophoresis and Southern blotting was performed with modifications as in (Bittmann et al., 2020) (link). For each timepoint, approximately 4x10 Afterwards, plugs were washed with TE (10 mM Tris-HCl pH 8.0, 1 mM EDTA) for 1-2 h each wash, placed into PK buffer (1 mg/ml sarcosyl, 0.5 M EDTA, 2 mg/ml proteinase K) and incubated at 55 °C for 2 days. Plugs were washed three more times with TE before use.
Plugs were loaded on a gel containing 1% agarose (Bio-Rad Cat. 1620138) in 0.5x TBE (45 mM Tris, 45 mM borate, 0.5 mM EDTA). Electrophoresis was carried out in 14 °C cold 0.5x TBE in a CHEF DR-III system (Bio-Rad, initial switch time 60 sec, final switch time 120 sec, 6 V/cm, angle 120°, 24 h). Afterwards, the gel was stained with 1 μg/ml ethidium-bromide in 0.5x TBE for one hour and de-stained with deionized water. Images were taken using a GenoSmart gel documentation system (VWR).
For Southern blotting, the DNA was nicked in 0.125 M HCl for 10 min, denatured in 1.5 M NaCl, 0.5 M NaOH for 30 min and neutralized by 0.5 M Tris, 1.5 M NaCl (pH 7.5) for 30 min.
The DNA was transferred onto a Hybond-N+ membrane (GE healthcare) and UV-cross-linked (Stratagene Stratalinker 1800, auto-crosslink function). The membrane was probed with a radioactive (α-32 P dCTP) labeled TRP1 fragment and imaged using a Typhoon FLA 9000 imaging system (GE Healthcare).
Plugs were loaded on a gel containing 1% agarose (Bio-Rad Cat. 1620138) in 0.5x TBE (45 mM Tris, 45 mM borate, 0.5 mM EDTA). Electrophoresis was carried out in 14 °C cold 0.5x TBE in a CHEF DR-III system (Bio-Rad, initial switch time 60 sec, final switch time 120 sec, 6 V/cm, angle 120°, 24 h). Afterwards, the gel was stained with 1 μg/ml ethidium-bromide in 0.5x TBE for one hour and de-stained with deionized water. Images were taken using a GenoSmart gel documentation system (VWR).
For Southern blotting, the DNA was nicked in 0.125 M HCl for 10 min, denatured in 1.5 M NaCl, 0.5 M NaOH for 30 min and neutralized by 0.5 M Tris, 1.5 M NaCl (pH 7.5) for 30 min.
The DNA was transferred onto a Hybond-N+ membrane (GE healthcare) and UV-cross-linked (Stratagene Stratalinker 1800, auto-crosslink function). The membrane was probed with a radioactive (α-32 P dCTP) labeled TRP1 fragment and imaged using a Typhoon FLA 9000 imaging system (GE Healthcare).
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