The following are the equipment used in our experiment: The sizes and ζ potential of IO-seeds, MNPs were measured by dynamic light scattering (JEM zetasizer Nano-ZS90, Malvern). The transmission electron microscopy (TEM) images of IO-seeds, IO@Si were obtained from a JEM-2100F electron microscope (JEOL, Japan). An 808-nm near-infrared (NIR) laser (ADR-1860, Shanghai, China) was applied for nano-LAMP experiment; meanwhile, the real-time temperature and thermal images were monitored by the infrared thermal camera (Fotric 225 s, Fotric Technology Company, Shanghai, China). A PCR System (ProFlex, Life Technology company Shanghai, China) was employed for conventional LAMP experiment.
Proflex
Manufactured by Thermo Fisher Scientific
Sourced in United States
The ProFlex is a thermal cycler designed for PCR amplification. It provides precise temperature control and is capable of performing various PCR protocols.
Automatically generated - may contain errors
Lab products found in correlation
Quantstudio 5 real time pcr system, by Thermo Fisher Scientific (4 mentions)
Purelink rna mini kit, by Thermo Fisher Scientific (2 mentions)
High capacity cdna reverse transcription kit, by Thermo Fisher Scientific (2 mentions)
Dlight, by Addgene (2 mentions)
Hchr2 mcherry, by Addgene (2 mentions)
Kapa hifi hotstart readymix, by Takara Bio (2 mentions)
T4 ligase, by New England Biolabs (2 mentions)
Omni ultracompetent bacterial cells, by Zymo Research (2 mentions)
Restriction enzyme, by New England Biolabs (2 mentions)
Nanodrop 1000, by Thermo Fisher Scientific (2 mentions)
Jem 2100f electron microscope, by JEOL (1 mentions)
2 propanol, by Sinopharm (1 mentions)
Trizol reagent, by Thermo Fisher Scientific (1 mentions)
Nebnext ultra rna library prep kit, by Illumina (1 mentions)
Hiseq 2500 platform, by Illumina (1 mentions)
Agilent bioanalyzer 2100 system, by Agilent Technologies (1 mentions)
Chloroform, by Sinopharm (1 mentions)
Agarose, by Biowest (1 mentions)
Sw41ti rotor, by Beckman Coulter (1 mentions)
Superscripttm 2 reverse transcriptase kit, by Thermo Fisher Scientific (1 mentions)
25 protocols using proflex
1
Characterization of IO-Seeds and MNPs
2
Characterization of SPINK13 in HCC
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An Agilent Bioanalyzer 2100 system (Agilent Technologies Inc., Santa Clara, CA, USA) was used for the determination of DNA concentration; Proflex (Life Technologies, Carlsbad, CA, USA) was used for polymerase chain reaction, and an Illumina Hiseq2500 platform (Illumina Inc., San Diego, USA) was used for high-throughput sequencing. TRIzol reagent was obtained from Invitrogen Corp. (Waltham, MA, USA); chloroform and 2-propanol were purchased from Sinopharm (Shanghai, China); Agarose was obtained from Biowest (Barcelona, Spain); a human Ribo-Zero Magnetic Gold Kit and NEBNext® Ultra™ RNA Library Prep Kit were purchased from Illumina Inc. (USA); Human hepatocellular carcinoma cell line, MHCC97-H cells, was donated from Dr. Feng Fan; and SPINK13 protein was synthesized by our laboratory from exogenous expression of HE239T cells. The study was conducted in accordance with the Declaration of Helsinki (as revised in 2013).
3
Heat Treatment and 70S Formation Assay
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For heat treated 50S subunits, 10 pmol 50S subunit was diluted in buffer A with 10 mM MgCl2 to a final volume of 20 μl. This mixture was then incubated at the desired temperature for 30 min on a ProFlex (ThermoFisher) PCR cycler with a heated lid and placed on the bench for 30 min to cool slowly to room temperature. For 70S formation assays, 10 pmol 50S subunit and 20 pmol WT untagged 30S subunit were diluted in buffer A with 15 mM MgCl2 to a final volume of 20 μl. These samples were then incubated at 37°C for 30 min. Samples were loaded onto a 15–40% sucrose gradient in buffer A with 10 or 15 mM MgCl2. Gradients were then placed in a SW-41 Ti rotor (Beckman-Coulter) and centrifuged at 29 000 rpm (100 000 × g) for 13 h. An ISCO gradient fractionation system was used to measure A254 traces.
4
S. agalactiae Atr Gene PCR Assay
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S. agalactiae specific 780 bp Atr gene (GenBank accession number: AF15135) were used as internal positive control for the PCR assay to confirm the identified isolates. The forward and reverse primer sequences CAA CGA TTC TCT CAG CTT TGT TAA and TAA GAA ATC TCT TGT GCG GAT TTC were used, respectively. The PCR reaction volume was 25 μL including 1 μL of bacterial DNA, 0.5 μL of forward primer, 0.5 μL of reverse primer, 12.5 μL of 2x Taq Premix-Master mix, and 10 μL of sterile double distilled water. Amplification of thermal cycles were as follows: an initial denaturation step for 5 min at 94°C, followed by 35 cycles of 94°C for 30 s, 55°C for 55 s, and 72°C for 1 min, and a final extension cycle of 72°C for 10 min using [12 (link)] Proflex, Thermofisher, USA. PCR products and 100-bp DNA size marker were run simultaneously on 1.5% agarose gel stained with DNA safe stain at 60 V for 1 hour. Finally, the agarose gel was visualized and photographed using UV Tech Cambridge.
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S. agalactiae specific 780 bp Atr gene (GenBank accession number: AF15135) were used as internal positive control for the PCR assay to confirm the identified isolates. The forward and reverse primer sequences CAA CGA TTC TCT CAG CTT TGT TAA and TAA GAA ATC TCT TGT GCG GAT TTC were used, respectively. The PCR reaction volume was 25 μL including 1 μL of bacterial DNA, 0.5 μL of forward primer, 0.5 μL of reverse primer, 12.5 μL of 2x Taq Premix-Master mix, and 10 μL of sterile double distilled water. Amplification of thermal cycles were as follows: an initial denaturation step for 5 min at 94°C, followed by 35 cycles of 94°C for 30 s, 55°C for 55 s, and 72°C for 1 min, and a final extension cycle of 72°C for 10 min using [12 (link)] Proflex, Thermofisher, USA. PCR products and 100-bp DNA size marker were run simultaneously on 1.5% agarose gel stained with DNA safe stain at 60 V for 1 hour. Finally, the agarose gel was visualized and photographed using UV Tech Cambridge.
5
Chondrocyte Gene Expression Analysis
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Total RNA was extracted from cultured primary human chondrocytes using a PureLink RNA Mini Kit (Ambion, Cat#12183018A) and 2-mercaptoethanol (Thermo Fisher Scientific, Cat#31350010). To obtain complementary DNA (cDNA), 50 ng RNA was reverse-transcribed via a High-Capacity cDNA Reverse Transcription Kit (Thermo Fisher Scientific, Cat#4368814) using a thermal cycler (ProFlex, Thermo Fisher Scientific) in accordance with the manufacturer’s instructions. For qRT-PCR analysis, all the genes were amplified using TaqMan® Gene Expression Assays for CHAD (Cat#4331182, Hs00154382_m1), HIF1α (Cat#4331182, Hs00153153_m1), COL2A1 (Cat#4331182, Hs00264051_m1), and internal control genes (housekeeping genes: actin beta (ACTB, Cat#4331182, Hs01060665_g1) in accordance with the manufacturer’s protocol [11 (link), 15 (link), 16 ]. Each gene was amplified using an RT-PCR reaction mix prepared with 1 μl TaqMan Gene Expression Assay, 10 ml of TaqMan Gene Expression Master Mix (Cat. #4369016), 4 ml of cDNA template, and UltraPure DNase/ RNase-Free distilled water (Cat. #10977035) on MicroAmp Fast Optical 96-well reaction plates (Cat. #4346906). The Applied Biosystems 7300/7500 RT-PCR system was used with the following reaction protocol: maintained at 50°C for 2 min, maintained at 95°C for 10 min, and alternating between 95°C for 15 s and at 60°C for 1 min and 40 cycles.
6
Genetic Confirmation by PCR
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The animal genotype was confirmed by polymerase chain reaction (PCR) using DNA extracted from the tail tips and specific primers under the conditions described in Table 1 . PCR was carried out in a ProFlex thermal cycler (Thermo Fisher Scientific), in 0.2 mL plastic tube strips containing 3 μL of genomic DNA, 2.63 μL of KAPA, 0.625 μL of 10 μM primer mix, and 6.25 μL of distilled water.
7
Comprehensive mRNA Extraction and qPCR Analysis
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Extraction of mRNA was conducted in three biological replicates using the RNeasy® Plus Mini Kit from Qiagen (Valencia, CA, USA) [35] (link), [36] (link). To obtain cDNA, 10 ng RNA was reverse transcribed using the SuperscriptTM II reverse transcriptase kit (200 U, Life Technologies, Inc.) and a PCR (ProFlex; Thermo Fisher Scientific, Inc.). Real-time PCR was performed using 2 μL cDNA, 0.4 μL for forward and reverse primers and following the instruction of PowerUpTM SYBR® Green Master Mix (Applied Biosystems, Austin, TX, USA) in a QuantStudio® 5 Real-Time PCR System (96-well, Thermo Fisher Scientific). Primers were designed with Primer3Plus (http://www.bioin forma tics.nl/cgi-bin/prime r3plu s/prime r3plu s.cgi) (Supplementary Table 5 ). The qPCR program was performed as previously described [35] (link), [36] (link). Ct recorded at 0.15 ΔRn and melt curve to verify PCR products. Data were analyzed using ΔΔCt method [37] (link) and were expressed as −ΔΔCt.
8
Quantification of Serum miRNAs by qRT-PCR
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For the validation experiments, the serum miRNA samples were reverse-transcribed using a miRCURY LNA RT Kit (Qiagen, Hilden, Germany), according to the manufacturer’s protocol, on a Proflex thermal cycler (Thermo Fisher Scientific, Waltham, USA). Then, the candidate miRNAs were quantified by qRT-PCR using specific primers and a miRCURY LNA SYBR Green PCR Kit (Qiagen, Germany). The samples were run on the qPCR plates in duplicate on the LightCycler 480 Real-Time PCR System (Roche, Switzerland). miR-103a-3p and miR-16-5p were used as endogenous control genes. The stability of the reference miRNAs was calculated by the NormFinder algorithm. Relative miRNAs expression was calculated using an efficiency corrected calculation model, based on multiple samples and multiple reference genes [40 ].
9
Genotyping LATS1 and CREBRF Founder Mice
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Total DNA was extracted from the toe/tail biopsy from 20 LATS1 and 6 CREBRF founder mice as described (Kolesar et al. 2013 (link)) and amplified with Taq DNA Polymerase (New England Biolabs) using the primer sets reported in Supplemental Material, Table S1 . The PCR was performed in a ProFlex thermal cycler (ThermoFisher Scientific) with the following PCR amplification profile: one cycle of 94° for 2 min; 35 cycles of 94° for 20 sec, 55° for 20 sec, and 68° for 60 sec; and one cycle of 68° for 5 min. The PCR products were cloned using the TOPO 4 kit with Escherichia coli DH5α cells (ThermoFisher Scientific). For each LATS1 or CREBRF founder mouse, 48–96 bacterial colonies were picked for colony PCR, amplified, and sequenced individually after PCR product purification with LATS1 Fw Primer and CREBRF Fw Primer 2, respectively (Figure S1 and Figure S2 ). The CREBRF blastocysts were amplified using the primer sets reported in Table S1 and Sanger-sequenced directly.
10
Polymerase Stop Assay for RHPS4 Evaluation
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Polymerase stop assay was performed as reported20 (link). Briefly, the experiment was conducted adding RHPS4 (0; 1; 2; 5; 10 µM) to a 25 µl reaction containing DreamTaq DNA Polymerase (ThermoFisher Scientific), 4.6 ng/reaction DNA, 0.2 mM dNTPs and 0.4 μM of each primer. The PCR was performed in a ProFlex thermal cycler (ThermoFisher Scientific) with the following PCR amplification profile: one cycle of 95 °C for 2 min; 30 cycles of 95 °C for 30 sec, 56 °C for 30 sec, and 72 °C for 30 sec. Products of amplification were run on a 2% agarose gel and detected with ethidium bromide fluorescence on a Bio-Rad ChemiDoc MP Imaging System (Bio-Rad, Carlsbad, CA). Quantitation was performed on three or more independent experiments.
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