The Isolink kit (Paul Scherrer Institut, Villigen, Switzerland) to prepare the labeling precursor ([99mTc(H2O)3 (CO)3]+) contained the following lyophilized ingredients: 8.5 mg sodium tartrate, 2.85 mg sodium tetraborate decahydrate, 7.15 mg of sodium carbonate and 4.5 mg sodium bicarbonate. Freshly eluted 99mTcO4− from a commercial General Electric Healthcare, UK generator (~2 GBq) in 1 mL saline buffer was added to the kit vial and incubated at 100°C for 20 min. The vial was then allowed to cool to RT and the solution neutralized by adding stepwise 1M HCl to pH ~7 and then checked on pH paper (Merck Millipore, Milan, Italy). The radiochemical purity (RCP) of the product was analyzed by high performance liquid chromatography (HPLC) and RPC was greater than 95%.
Ph paper
Manufactured by Merck Group
Sourced in Germany, United States
PH paper is a type of litmus paper used to measure the pH level of a solution. It is a simple and inexpensive tool for quickly determining the acidity or basicity of a liquid. PH paper changes color depending on the pH of the solution it is dipped in, providing a visual indication of the pH value.
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Lab products found in correlation
Itlc sg strip, by Agilent Technologies (1 mentions)
Nap 25, by GE Healthcare (1 mentions)
Drx 500 mhz spectrometer, by Bruker (1 mentions)
Mercury400 mhz, by Bruker (1 mentions)
Ph meter, by Mettler Toledo (1 mentions)
Nmr tube, by Wilmad (1 mentions)
Dntp mix, by Tiangen Biotech (1 mentions)
Rtx reverse transcriptase, by New England Biolabs (1 mentions)
Bst 2.0 warmstart dna polymerase, by New England Biolabs (1 mentions)
Isothermal amplification buffer, by New England Biolabs (1 mentions)
Bst 3.0 dna polymerase, by New England Biolabs (1 mentions)
Warmstart rtx reverse transcriptase, by New England Biolabs (1 mentions)
8 protocols using ph paper
1
Preparation of [99mTc(H2O)3(CO)3]+ Labeling Precursor
2
Radioiodine Labeling Protocol
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For patient release, different release criteria are set regarding the final activity, RCY and RCP. Intermediate activity measurements during- and after labelling are performed using the dose calibrator with validated geometries. To determine the RCY, radio-(I)TLC is used. An ITLC-SG strip [0.5 × 10 cm, dried 20 min at 160 °C from Agilent Technologies (Folsom, CA, United States) is spotted with 3 µL labelling solution, at 1 cm from the bottom of the strip and is dried for 30 s before placement in 250 µL 1 M Sodium-Citrate solution (pH 5). After separation, the strip is dried for 10 min, thereafter sealed in parafilm. For measurements, standardized settings for the drop point, endpoint, scanning time and regions of interest (ROI) for the ITLC-SG strip are used. The ITLC strip is cut at a retention factor (RF) of 0.7, each part is measured in the gamma counter. The pH is determined using pH paper from Merck (Darmstadt, Germany). An HPLC is prepared with and activity of 6 MBq/100 µL and was injected at time points t = 0 (directly after labelling), 4, 24 and 48 h, analysis is performed according to the earlier described settings.
3
Radiochemical Characterization of PET Tracers
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The appearance test was performed visually by checking the product vial for particles or turbulence in the final volume. The pH of the product was determined using Merck pH paper (pH 4–9). The radiochemical purity, radiochemical identity, and radiochemical stability of [11C]flumazenil and [11C]L-deprenyl were analyzed by Agilent analytical high-performance liquid chromatography (HPLC). This HPLC is equipped with a quaternary pump G1311A, manual injector, ultraviolet (UV) detector G1314D, and radio detector. An analytical HPLC column from Xbridge (C18, 4.6 × 150 mm, 5 µm particle size) was used, and an isocratic mobile phase of 25% of acetonitrile and 75% of 10 mM phosphoric acid was used for [11C]flumazenil. A mobile phase of 50% acetonitrile and 50% ammonium phosphate buffer pH–9 was used for [11C]L-deprenyl. The flow rate was 2 mL/min. The identification of products was determined by co-injecting with the corresponding reference standard.
4
Melamine Detection in Milk via SERS
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The milk samples with 0.5 % fat content (Dansk Maelk, Jacobs Douwe Egberts Professional, København, Denmark) were purchased from a local commercial supplier. The samples, spiked with melamine, were pretreated prior to SERS measurement, using a gel filtration column (NAP-25, GE Healthcare, Pittsburgh, PA, USA) as also described by A. Kim et al. 61 (link) In short, 2.5 ml milk samples, spiked with various melamine concentrations (0.3 ppm -2.5 ppm) using the pH 7.4 stock solutions, were loaded into the saturated filtration column. The milk was allowed to sink into the column and subsequently eluted in 2.5 ml fractions with PBS pH 7.4. For SERS measurements the pH of the fractions were adjusted with 10 % HCl to a pH value between 3 to 4, which was verified with pH paper (Merck KGaA, Darmstadt, Germany).
5
Zinc Acetate Concentration and Purification
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A stock solution of 160 mM Zn(CH3CO2)2 was prepared, then diluted to a final concentration of 3.2 mM. A stock solution of ammonium hydroxide at 1 M was also prepared. Sample was run through a C18 column at a flow rate of 0.5 ml/min. Before electrospray, a neutralizing solution of 1 M ammonium hydroxide was added at a flow rate of 5 μl/min, then the solution of 3.2 mM zinc acetate was added at a flow rate of 5 μl/min. Post-LC pH was verified by collecting the flow through and spotting on pH paper (Sigma).
6
NMR Characterization of 2OX Degradation
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1H NMR spectra were recorded on either a Varian Mercury 400 MHz or Bruker DRX 500 MHz spectrometer (Bruker, Massachusetts, USA). Diffusion Ordered Spectroscopy (DOSY) experiments were performed using a longitudinal encode-decode (LED) pulse sequence with a 5–95% varying gradient strength over 19 points.
2OX was dissolved in buffers adjusted to a known pH (+/− 0.2 pH units) as measured using either pH paper (Sigma-Aldrich, Missouri, USA) or a pH meter (Mettler Toledo, Ohio, USA). Buffers used were 0.5 M pH 5 sodium acetate, pH 7 sodium phosphate or pH 9 sodium carbonate. Buffers were prepared at the appropriate pH in water, then exchanged 3 × in 99.8% D2O under vacuum. Reactions were held at constant temperature in a thermostatted oven for periods of up to 6 months.
NMR measurements of 2OX were recorded starting from concentrations of 2, 20 or 200 mM using ~ 0.5 mL volumes in Wilmad NMR tubes (Wilmad Lab Glass, New Jersey, USA) capped under air. Samples were measured periodically, and the kinetics measured by comparison of proton integrations. Results reported in Figure SI4 were conducted at 25° C or 40° C as noted.
2OX was dissolved in buffers adjusted to a known pH (+/− 0.2 pH units) as measured using either pH paper (Sigma-Aldrich, Missouri, USA) or a pH meter (Mettler Toledo, Ohio, USA). Buffers used were 0.5 M pH 5 sodium acetate, pH 7 sodium phosphate or pH 9 sodium carbonate. Buffers were prepared at the appropriate pH in water, then exchanged 3 × in 99.8% D2O under vacuum. Reactions were held at constant temperature in a thermostatted oven for periods of up to 6 months.
NMR measurements of 2OX were recorded starting from concentrations of 2, 20 or 200 mM using ~ 0.5 mL volumes in Wilmad NMR tubes (Wilmad Lab Glass, New Jersey, USA) capped under air. Samples were measured periodically, and the kinetics measured by comparison of proton integrations. Results reported in Figure SI4 were conducted at 25° C or 40° C as noted.
7
RT-LAMP Assay for Rapid Detection
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RT-LAMP assays were assembled in a total reaction volume of 20 μl, each 10 μl iLACO reactions contained: 2 μl of home-made 10X buffer (100 mM (NH4)2SO4, 500 mM KCl, 80 mM MgSO4, and 1% v/v Tween-20), 2.8 μl of dNTP mix (10mM each, TianGen), l μl of Bst 2.0 WarmStart DNA Polymerase (New England Biolabs) or Bst V7.16, 0.5 μl of RTx reverse transcriptase (New England Biolabs) or home-made RT, 1 μl of a combination of dyes (both 0.5 μl of phenol red and azure II, 2mM of each dye), and above components were mixed in DEPC H2O up to 10 μl, then adjusted pH to 7.5 with 1M KOH and measured by pH paper (Supelco). In addition to the above, 2 μl of 10X LAMP primer mix (2 μM F3, 2 μM B3, 16 μM FIP, 16 μM BIP, 4 μM LF and 4 μM LB), 1 μl sample and DEPC H2O up to 20 μl were required.
8
Evaluation of iLACO-dual for SARS-CoV-2 and H1N1 Detection
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We first tested the performance of iLACO-dual with in vitro transcribed virus RNA fragments for SARS-CoV-2 and 2009 H1N1 influenza virus [7 , 30 ]. When the RT-LAMP assays were performed with Bst 2.0 WarmStart DNA polymerase, each reaction condition (20 μL) consisted of 2 μL Isothermal Amplification Buffer (New England Biolabs), 2.8 μL dNTP Mix (10 mM), 0.5 μL WarmStart RTx Reverse Transcriptase (New England Biolabs), 1 μL Bst 2.0 WarmStart DNA polymerase, 2 μL RT-LAMP primer mix (0.8 μM FIP, 0.8 μM Q-FIP: Fd, 1.6 μM BIP, 0.2 μM F3/B3, 0.4 μM LF/LB), 1 μL RNA mix of SARS-CoV-2 and H1N1, 10.7 μL DEPC water. Bst 3.0 DNA polymerase (New England Biolabs, USA) was the same as Bst 2.0 WarmStart DNA polymerase, except the KCl is 150 mM in Isothermal Amplification Buffer instead of 50 mM for Bst 2.0.
For homemade Bst variant Bst v7.16, the Isothermal Amplification Buffer contains 10 mM (NH4)2SO4, 50 mM KCl, 8 mM MgSO4, 0.1% Tween 20, and all components were mixed in DEPC water, then adjusted pH to 7.5 with 1M KOH and measured by pH paper (Supelco). The other reagents of the reaction condition (20 μL) were the same as Bst 2.0 and Bst 3.0 DNA polymerase.
For homemade Bst variant Bst v7.16, the Isothermal Amplification Buffer contains 10 mM (NH4)2SO4, 50 mM KCl, 8 mM MgSO4, 0.1% Tween 20, and all components were mixed in DEPC water, then adjusted pH to 7.5 with 1M KOH and measured by pH paper (Supelco). The other reagents of the reaction condition (20 μL) were the same as Bst 2.0 and Bst 3.0 DNA polymerase.
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