LitCovid-sentences  

PMC:7143804 / 38489-43081 JSONTXT 10 Projects

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Id Subject Object Predicate Lexical cue
T298 0-4 Sentence denotes 3.5.
T299 5-22 Sentence denotes DNA Amplification
T300 23-211 Sentence denotes First, to determine the temperature window of operation, MDA reactions are performed at 25 °C and 30 °C using the Illustra GenomiPhi V2 DNA amplification kit and EvaGreen fluorescence dye.
T301 212-361 Sentence denotes From the literature, we know that this reaction does not work above 35 °C due to degradation of the protein activity in presence of a substrate [44].
T302 362-508 Sentence denotes In Figure 13, a graph of the fluorescence signal during MDA reactions at 25 °C and 30 °C, together with their non template control (NTC) is shown.
T303 509-884 Sentence denotes These reactions are carried out in a conventional Bio-Rad CFX96 Touch Real-Time PCR machine (Bio-Rad Laboratories, Inc., Hercules, CA, USA) and the results show that the chosen proof-of-principle DNA amplification reaction is temperature dependent to some extent, but that there is a wide range of temperatures at which the amplification can be performed, i.e., 25°C to 35°C.
T304 885-1000 Sentence denotes This makes the functioning of the integrated resistive heater less critical than the stability shown in Figure 11c.
T305 1001-1203 Sentence denotes MDA reactions are also performed inside an Eppendorf tube (Eppendorf AG, Hamburg, Germany) and inside the chip, again using the Illustra GenomiPhi V2 DNA amplification kit and EvaGreen fluorescence dye.
T306 1204-1318 Sentence denotes As heat supply the water bath of an IKA Rotary Evaporator RV 8V (IKA-Werke, Staufen im Breisgau, Germany) is used.
T307 1319-1422 Sentence denotes This water bath is according to its specification stable within a range of the set temperature ±0.1 °C.
T308 1423-1547 Sentence denotes The chip and an Eppendorf tube are loaded with the reaction mixture containing the DNA sample and the EvaGreen dye solution.
T309 1548-1681 Sentence denotes Here, the Eppendorf tube is serving as a control to show that the fabrication steps of the chips are not inhibiting the MDA reaction.
T310 1682-1772 Sentence denotes The inlet and outlet of the chip are sealed with the Microseal “B” PCR plate sealing foil.
T311 1773-1909 Sentence denotes The closed chip and tube are heated up in a separate water bath to 95 °C and kept at that temperature for 3 min to denaturate the dsDNA.
T312 1910-2044 Sentence denotes Subsequently, the chip and tube are cooled down by placing it in an ice bath for 5 min after which the rest of the reagents are added.
T313 2045-2107 Sentence denotes The complete mixtures are according to Table A3 in Appendix D.
T314 2108-2281 Sentence denotes After closing the chip and tube again, they are placed in the water bath of the rotary evaporater and left there for 90 min, after which the reaction is terminated at 65 °C.
T315 2282-2392 Sentence denotes The MDA is also performed inside the chip, but with the integrated Au resistive heater serving as heat source.
T316 2393-2437 Sentence denotes The set up shown schematically in Figure 12.
T317 2438-2504 Sentence denotes The same procedure is followed as with the water bath heated test.
T318 2505-2551 Sentence denotes Denaturation is done in a separate water bath.
T319 2552-2775 Sentence denotes The heater is driven by an input potential of 3.2 V to get to a temperature of 30 °C and at the end of the reaction, the system is heated up to 65 °C by applying a potential of 9.2 V in order to terminate the amplification.
T320 2776-2846 Sentence denotes In Figure 14 the logged temperature during the amplification is shown.
T321 2847-3037 Sentence denotes After the amplifications, the reaction mixtures are pipetted out of the chips and tubes and into 1 mL quartz cuvettes containing 55 μL MilliQ DI water (Merck Millipore, Burlington, MA, USA).
T322 3038-3155 Sentence denotes Fluorescence measurements are done in a Horiba Scientific FluoroMax+ spectrofluorometer to verify each amplification.
T323 3156-3331 Sentence denotes The mixture is excitated at a wavelength of 500 nm and the emission spectrum is measured at wavelengths from 510 nm to 550 nm (bounded EvaGreen dye has a peak at 525 nm [63]).
T324 3332-3492 Sentence denotes The measured spectra are normalized by subtracting the background signal of a mixture containing only the reaction buffer, the sample buffer, EvaGreen, and DNA.
T325 3493-3574 Sentence denotes No Enzyme was added to this mixture, therefore no amplification could take place.
T326 3575-3646 Sentence denotes See Figure 15 for the results obtained in the Eppendorf tube and chips.
T327 3647-3741 Sentence denotes Figure A4 in Appendix E shows the background signal which is subtracted from all measurements.
T328 3742-4017 Sentence denotes As can be seen in Figure 15, the spectra of the amplification performed inside the chip, and by applying heat with the water bath as well as with the integrated Au-resistive heater, show the same trend as the amplification performed in the Eppendorf and heated by water bath.
T329 4018-4087 Sentence denotes There is an order of magnitude difference in the fluorescence signal.
T330 4088-4180 Sentence denotes However, the fluorescence intensity cannot be used as a value to quantify the amount of DNA.
T331 4181-4321 Sentence denotes EvaGreen is a bis-intercalating cyanine fluorescence dye consisting of two monomeric DNA-binding dyes which are linked by a flexible spacer.
T332 4322-4464 Sentence denotes These two DNA-binding dyes bind each in between two base pairs, which make them simple and fast, but also nonuniform and non-specific [44,63].
T333 4465-4592 Sentence denotes However, with this dye, a simple yes-or-no answer can be obtained if the amplification took place, as can be seen in Figure 15.