Oligonucleotides (Tn5ME-A, Tn5ME-B, Tn5MErev) were resuspended in oligo annealing buffer (10 mM Tris−HCl pH 8.0 (Invitrogen, 15568-025), 50 mM NaCl (Invitrogen, AM9759), 1 mM EDTA (Invitrogen, AM9260G)) to a final concentration of 100μM each. Equimolar amounts of Tn5MErev/Tn5ME-A and Tn5MErev/Tn5ME-B were mixed in separate 200 μl PCR tubes. Then, the adaptors were annealed on the PCR machine with the following PCR program (95°C for 5 min first, then the temperature was slowly ramped down to 25°C with the rate of −0.1°C/s, 25°C for 5 min). The Tn5 transposase was assembled with the following components: 0.04 vol. Tn5MErev/Tn5ME-A, 0.04 vol Tn5MErev/Tn5ME-B, 0.4 vol. 100% glycerol (Sigma-Aldrich, G9012), 0.3048 vol 2× dialysis buffer (100 mM HEPES−KOH (HEPES: Sigma-Aldrich, H3375; KOH: Sigma-Aldrich, 484016) at pH 7.2, 0.2 M NaCl (Invitrogen, AM9759), 0.2 mM EDTA (Invitrogen, AM9260G), 2 mM DTT (Thermo Fisher Scientific, 20291), 0.2% Triton X-100 (Sigma-Aldrich, T8787), 20% glycerol (Sigma-Aldrich, G9012)), 0.043 vol. pure Tn5 (46.55 μM), 0.1722 vol. water (Invitrogen, AM9932). The reagents were mixed thoroughly but gently, and the mixture was left on the bench at RT for 1 h to allow annealing of oligos to Tn5.
G9012
Manufactured by Merck Group
Sourced in United States
The G9012 is a Magnetic Stirrer Hotplate from the Merck Group. It is a laboratory equipment device used for mixing and heating solutions simultaneously. The device features a magnetic stirring mechanism and a built-in heating element to control the temperature of the samples.
Automatically generated - may contain errors
Lab products found in correlation
Am9260g, by Thermo Fisher Scientific (6 mentions)
Am9759, by Thermo Fisher Scientific (3 mentions)
H3375, by Merck Group (3 mentions)
Am9932, by Thermo Fisher Scientific (3 mentions)
T8787, by Merck Group (3 mentions)
Cm1135, by Thermo Fisher Scientific (2 mentions)
Maldi tof biotyper system, by Bruker (2 mentions)
Lsm 880, by Zeiss (2 mentions)
Methyl β cyclodextrin, by Tokyo Chemical Industry (1 mentions)
γ cyclodextrin, by Fujifilm (1 mentions)
Leucine, by Merck Group (1 mentions)
Imagej, by Zeiss (1 mentions)
Zen software, by Zeiss (1 mentions)
Macconkey agar, by Biolife (1 mentions)
Cm0469, by Thermo Fisher Scientific (1 mentions)
P4864, by Merck Group (1 mentions)
E10521, by Merck Group (1 mentions)
Propidium iodide, by Merck Group (1 mentions)
Sp 1120, by Vector Laboratories (1 mentions)
11 protocols using g9012
1
Tn5 Transposome Assembly Protocol
2
Tn5 Transposome Preparation Protocol
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Oligonucleotides (T7-Tn5ME, Tn5MErev) were resuspended in oligo annealing buffer (10 mM Tris−HCl pH 8.0 (Invitrogen, 15568-025), 50 mM NaCl (Invitrogen, AM9759), 1 mM EDTA (Invitrogen, AM9260G)) to a final concentration of 100 μM each. Equimolar amounts of Tn5MErev/T7-Tn5ME were mixed in separate 200 μl PCR tubes. Then, the adaptors were annealed on the PCR machine with the following PCR program (95°C for 5 min first, then the temperature was slowly ramped down to 25°C with the rate of −0.1°C/s, finally 25°C for 5 min). The T7−pA−Tn5 transposase was assembled with the following components: 0.08 vol. Tn5MErev/T7-Tn5ME, 0.4 vol. glycerol (Sigma-Aldrich, G9012), 0.3116 vol. 2× dialysis buffer (100 mM HEPES−KOH (HEPES: Sigma-Aldrich, H3375; KOH: Sigma-Aldrich, 484016) at pH 7.2, 0.2 M NaCl (Invitrogen, AM9759), 0.2 mM EDTA (Invitrogen, AM9260G), 2 mM DTT (Thermo Fisher scientific, 20291), 0.2% Triton X-100 (Sigma-Aldrich, T8787), 20% glycerol (Sigma-Aldrich, G9012)), 0.0362 vol. pure pA–Tn5 (55.55 μM), 0.1722 vol. water (Invitrogen, AM9932). The reagents were mixed thoroughly but gently, and the mixture was left on the bench at RT for 1 h to allow annealing of oligos to pA–Tn5.
3
Brain Tissue Clearing with ScaleS Solutions
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The schedule for tissue clearing with ScaleSF is described in Figure 2 A. Brain slices were permeabilized with ScaleS0 solution for 2 hr at 37°C, washed twice with PBS(–) (27575-31, Nacalai Tesque) for 15 min at 20–25°C, and cleared with ScaleS4 solution for 8–12 hr at 37°C. We treated brain slices with ScaleS4 solution for 12 hr in the data shown in this paper. The formula for ScaleS0 solution was 20% (w/v) sorbitol (06286-55, Nacalai Tesque), 5% (w/v) glycerol (G9012, Sigma-Aldrich), 1 mM methyl-β-cyclodextrin (M1356, Tokyo Chemical Industry), 1 mM γ-cyclodextrin (037-10643, Wako Pure Chemical Industries), and 3% (v/v) dimethyl sulfoxide (DMSO) (13407-45, Nacalai Tesque) in PBS(–), and that for ScaleS4 solution was 40% (w/v) sorbitol, 10% (w/v) glycerol, 4 M urea (35940-65, Nacalai Tesque), 0.2% (w/v) Triton X-100 (35501-15, Nacalai Tesque), and 25% (v/v) DMSO in distilled deionized water (DDW) (Miyawaki et al., 2016 (link)).
The optical clearing methods of brain slices with CUBIC, PACT, ScaleSQ(0) and SeeDB followed the protocol as below. Considering 1-mm-thick slices clearing, incubation time of each method was adjusted accordingly.
The optical clearing methods of brain slices with CUBIC, PACT, ScaleSQ(0) and SeeDB followed the protocol as below. Considering 1-mm-thick slices clearing, incubation time of each method was adjusted accordingly.
4
Cultivation of Mycobacterium tuberculosis Auxotrophs
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Mycobacterium tuberculosis H37Rv (ATCC #27294) was grown in 7H9 media supplemented with 10% oleic albumin dextrose catalase (OADC) (Remel, Thermo-Fisher) and 5% glycerol (G9012, Sigma- Aldrich). The auxotroph mutants of Mtb H37Rv 6206 (ΔpanCD, ΔleuC) and 6230 (ΔRD1, ΔpanCD) were kindly provided by Dr. William Jacobs (Sampson et al., 2004 (link); Sambandamurthy et al., 2006 (link)). 6230 was grown in the 7H9 supplemented with 0.05% tyloxapol (T0307, Sigma-Aldrich), 0.2% casamino acids (223050, BD BactoTM) and 24 μg/ml pantothenate (D -pantothenic acid hemicalcium salt, Sigma-Aldrich). The media for 6206 was similar to that used for 6230 with an additional supplement of 80 mg/ml leucine (Sigma-Aldrich).
5
Freeze-Dried Pathogen Storage and Propagation
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The food-borne pathogens that were selected for the study were E.
coli MTCC 443, and L. monocytogenes MTCC 657. The
strains were procured from MTCC, India. The samples were received as
freeze-dried cultured in glass vials. Upon receiving the samples, the cultures
were sub-cultured by inoculating the samples into 10 mL of sterile Tryptone Soya
Broth (TSB, HiMedia M641, Chennai, India) and incubating for 24 h at
37°C. After the incubation period, sterile 50% glycerol on
deionized water was added with the cultures at 1:1 ratio to make a final
concentration of 25% glycerol (Sigma Aldrich, G9012) in the glycerol
culture stocks. The samples were transferred to cryovials and were frozen at
−80°C until further use.
coli MTCC 443, and L. monocytogenes MTCC 657. The
strains were procured from MTCC, India. The samples were received as
freeze-dried cultured in glass vials. Upon receiving the samples, the cultures
were sub-cultured by inoculating the samples into 10 mL of sterile Tryptone Soya
Broth (TSB, HiMedia M641, Chennai, India) and incubating for 24 h at
37°C. After the incubation period, sterile 50% glycerol on
deionized water was added with the cultures at 1:1 ratio to make a final
concentration of 25% glycerol (Sigma Aldrich, G9012) in the glycerol
culture stocks. The samples were transferred to cryovials and were frozen at
−80°C until further use.
6
Antennal Autofluorescence Imaging in Chrysomela populi
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Antennae were dissected from cold anesthetized adults of C. populi (n = 4), briefly cleaned in an Ultrasonic Cleaner (VWRTM) and mounted in glycerol (G9012 Sigma-Aldrich) in two cover slips, allowing microscopic observation of the distal antennal tip from both sides. Autofluorescence scanning images were acquired on a confocal laser scanning microscope (LSM 880, Zeiss, Oberkochen, Germany) using a 40×/1.2 W C-Apochromat. Excitation of cuticular autofluorescence was conducted by a 405 nm laser diode and the fitting main beam splitter 405. Emission was detected in Lambda mode between 410 and 695 nm to discriminate between high and low concentrations of resilin or chitin, respectively. Approximately 300 scans at a thickness of 0.5 μm were employed to image the terminal (9th) flagellomere of the antennae from both sides. Maximum intensity projections were used for visualization of the entire image stacks. Images were processed using ZEN software (Zeiss) and ImageJ (Schneider et al., 2012 (link)).
7
Tn5 Transposome Assembly Protocol
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Oligonucleotides (Tn5ME-A, Tn5ME-B, Tn5MErev) were resuspended in oligo annealing buffer (10 mM Tris–HCl pH 8.0 (Invitrogen, 15568-025), 50 mM NaCl (Invitrogen, AM9759), 1 mM EDTA (Invitrogen, AM9260G)) to a final concentration of 100 μM each. Equimolar amounts of Tn5MErev/Tn5ME-A and Tn5MErev/Tn5ME-B were mixed in separate 200 μl PCR tubes. Then, the adaptors were annealed on the PCR machine with the following PCR program (95°C for 5 min first, then the temperature was slowly ramped down to 25°C with the rate of −0.1°C/s, 25°C for 5 min). The pA–Tn5 transposase was assembled with the following components: 0.04 vol. Tn5MErev/Tn5ME-A, 0.04 vol Tn5MErev/Tn5ME-B, 0.4 vol. 100% glycerol (Sigma-Aldrich, G9012), 0.3116 vol. 2× dialysis buffer (100 mM HEPES−KOH (HEPES: Sigma-Aldrich, H3375; KOH: Sigma-Aldrich, 484016) at pH 7.2, 0.2 M NaCl (Invitrogen, AM9759), 0.2 mM EDTA (Invitrogen, AM9260G), 2 mM DTT (Thermo Fisher scientific, 20291), 0.2% Triton X-100 (Sigma-Aldrich, T8787), 20% glycerol (Sigma-Aldrich, G9012)), 0.0362 vol pure pA–Tn5 (55.55 μM), 0.1722 vol water (Invitrogen, AM9932). The reagents were mixed thoroughly but gently, and the mixture was left on the bench at RT for 1 h to allow annealing of oligos to pA–Tn5.
8
Isolation and Identification of Respiratory Pathogens
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Clinical specimens were cultured aseptically onto blood agar (Columbia agar with 5% Sheep blood, 254,005 BD), chocolate agar (GC II agar with IsoVitaleX, 254,060, BD or blood Agar No. 2 Base, 257,011, BD) and MacConkey agar (4,016,702, Biolife Italiana Srl) and incubated at 35–37 °C in aerobic conditions with 5–10% CO2 for 24–48 h to address the isolation of respiratory bacterial pathogens.
Identification of isolates for respiratory pathogens was carried out by matrix assisted laser desorption ionization-time of flight (MALDI-TOF Biotyper System, Bruker Daltonics, Bremen, Germany) as previously described [13 (link)]. Actinobacillus pleuropneumoniae was also confirmed by PCR technique due to limitation of MALDI-TOF for the Actinobacillus genus [57 (link)]. Individual isolates were stored at -80 °C in brain heart infusion (CM1135, Oxoid) with 30% of glycerol (G9012, Sigma-Aldrich).
Identification of isolates for respiratory pathogens was carried out by matrix assisted laser desorption ionization-time of flight (MALDI-TOF Biotyper System, Bruker Daltonics, Bremen, Germany) as previously described [13 (link)]. Actinobacillus pleuropneumoniae was also confirmed by PCR technique due to limitation of MALDI-TOF for the Actinobacillus genus [57 (link)]. Individual isolates were stored at -80 °C in brain heart infusion (CM1135, Oxoid) with 30% of glycerol (G9012, Sigma-Aldrich).
9
Isolation and Identification of Respiratory and Digestive Pathogens
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Clinical specimens were cultured aseptically onto blood agar (Columbia agar with 5% Sheep blood, 254005 BD), chocolate agar (GC II agar with IsoVitaleX, 254060, BD or blood Agar No. 2 Base, 257011, BD) and MacConkey agar (4016702, Biolife Italiana Srl) and incubated at 35 ± 2 °C in aerobic conditions with 5–10% CO2 for 24–48 h to address the isolation of respiratory and systemic pathogens. Finally, for the isolation of digestive pathogens, specimens were cultured aseptically onto Blood agar, MacConkey agar and Xylose-Lysine-Desoxycholate Agar (XLD, CM0469, Oxoid). The plates were incubated at 35 ± 2 °C in aerobic conditions for 24 h.
Identification of isolates for respiratory pathogens (APP, P. multocida and B. bronchiseptica), S. suis and digestive pathogens was carried out by matrix-assisted laser desorption ionization-time of flight (MALDI-TOF Biotyper System, Bruker Daltonics, Bremen, Germany) as previously described [17 (link)]. Individual strains were stored at −80 °C in brain heart infusion (CM1135, Oxoid) with 30% of glycerol (G9012, Sigma-aldrich, St. Louis, MO, USA).
Identification of isolates for respiratory pathogens (APP, P. multocida and B. bronchiseptica), S. suis and digestive pathogens was carried out by matrix-assisted laser desorption ionization-time of flight (MALDI-TOF Biotyper System, Bruker Daltonics, Bremen, Germany) as previously described [17 (link)]. Individual strains were stored at −80 °C in brain heart infusion (CM1135, Oxoid) with 30% of glycerol (G9012, Sigma-aldrich, St. Louis, MO, USA).
10
Propidium Iodide Labeling of Injured Spinal Cord
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To reveal the dying spinal cord cells after injury, animals were deeply anesthetized with 0.3-0.5% tricaine methane sulfonate (MS-222; E10521, Sigma-Aldrich) and dissected in ice-cold extracellular solution (see “Electrophysiological recordings” section). Axial muscles and vertebral arches were removed to expose and facilitate the permeation in the spinal cord tissue. Then, the spinal cord preparations were transferred in an extracellular solution containing 5% PI (Propidium Iodide; P4864, Sigma-Aldrich) and incubated at room temperature for 30 min with gentle agitation. The tissue was then rinsed three times for 10 min in fresh extracellular solution and fixed in 4% PFA overnight at 4 °C. After thorough rinses with PBS; tissues were mounted on glass slides in an 80% glycerol (G9012, Sigma-Aldrich) in PBS mounting solution. In some experiments (Supplementary Fig. 1 ), spinal MNs were first traced as described above (see “Spinal motoneuron tracing” section) and then processed for PI labeling.
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