For untargeted GC–MS analysis, the standard Kundu et al. method was followed [26 (link)]. An extract of 480 µL of pure methanol along with 20 µL of 0.2 mg mL−1 ribitol (adonitol) as internal standard was added to 20 mg of dried callus and shoot sample. The mixture was vigorously shaken for 2 min and then heated at 70 °C for 15 min (ThermoStatC, Eppendorf, Hamburg, Germany). To this, an equal volume of water was added and vortexed (Spinix vortex shaker, Tarsons, Mumbai, India), followed by the addition of 250 µL of chloroform and thoroughly mixed. This mixture was centrifuged (Eppendorf R centrifuge 5430 R) at 2200× g for 10 min at room temperature (~22–25 °C). The upper aqueous phase was pipetted out and dried in a speed vacuum rotator (Concentrator plus, Eppendorf) at 45 °C for 2.5 h. The dried fraction was redissolved in 40 µL of 20 mg mL−1 methoxamine hydrochloride prepared in pyridine and then incubated for 90 min at 30 °C (ThermoStatC, Eppendorf). A total of 60 µL of MSTFA (N-methylN-(trimethylsilyl) trifluroacetamide) was added to the above solution and incubated for 30 min at 37 °C. A total of 100 µL of this derivatized sample was transferred in an insert containing a GC–MS glass vial and stored at 4 °C until it was analyzed in GC–MS/MS (TQ8050 NX, Shimadzu, Kyoto, Japan).
Spinix vortex shaker
Manufactured by Tarsons
Sourced in India
The Spinix vortex shaker is a laboratory instrument used to mix and agitate samples in test tubes or microplates. It provides a consistent vortex motion to ensure thorough mixing of the contents.
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4 protocols using spinix vortex shaker
1
Untargeted GC-MS Metabolite Analysis
2
Extraction and Analysis of Antimicrobials from Soil
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The antimicrobials
were extracted
from soil samples by dissolving the soil in methanol (1:3 w/v) and
kept for overnight shaking (Genei Rocker-100, Bangalore, India). Briefly,
10 g of solid was weighed and dissolved in 30 mL of methanol. The
soil was dissolved thoroughly by mixing on a vortex shaker (Spinix
vortex shaker, Tarsons, Mumbai, India) for 10 min followed by overnight
shaking. After that, soil samples were subjected to centrifugation
at rcf 13 752g for 30 min. Approximately,
30 mL of organic solvent was collected from each soil sample and transferred
to six glass, Vensil, tubes (5 mL each) for drying using nitrogen
gas (TurboVap). Post drying, 500 μL of methanol was added to
all Vensil tubes, vortexed, and mixed for 5 min. Finally, concentrated
samples were dried and reconstituted with 200 μL of methanol.
To remove the presence of any particulate matter that may contaminate
the LC-MS/MS system, reconstituted samples were transferred into a
1.5 mL microcentrifuge tube and subjected to centrifugation at rcf
16 502g for 30 min. After that, 50 μL
of the supernatant was carefully collected and transferred into LC-MS/MS
vials. From this, 10 μL of the sample was injected into the
mass spectrometer for analysis. We have received 29 soil samples,
and analyte estimates are given inTable 8 .
were extracted
from soil samples by dissolving the soil in methanol (1:3 w/v) and
kept for overnight shaking (Genei Rocker-100, Bangalore, India). Briefly,
10 g of solid was weighed and dissolved in 30 mL of methanol. The
soil was dissolved thoroughly by mixing on a vortex shaker (Spinix
vortex shaker, Tarsons, Mumbai, India) for 10 min followed by overnight
shaking. After that, soil samples were subjected to centrifugation
at rcf 13 752g for 30 min. Approximately,
30 mL of organic solvent was collected from each soil sample and transferred
to six glass, Vensil, tubes (5 mL each) for drying using nitrogen
gas (TurboVap). Post drying, 500 μL of methanol was added to
all Vensil tubes, vortexed, and mixed for 5 min. Finally, concentrated
samples were dried and reconstituted with 200 μL of methanol.
To remove the presence of any particulate matter that may contaminate
the LC-MS/MS system, reconstituted samples were transferred into a
1.5 mL microcentrifuge tube and subjected to centrifugation at rcf
16 502g for 30 min. After that, 50 μL
of the supernatant was carefully collected and transferred into LC-MS/MS
vials. From this, 10 μL of the sample was injected into the
mass spectrometer for analysis. We have received 29 soil samples,
and analyte estimates are given in
3
Genomic DNA Extraction from Oocysts
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Total genomic DNA was isolated using a QIAamp DNA Stool mini kit (Qiagen, Germany) as per the manufacturer's protocol with some modifications from (i) oocysts purified as described above or (ii) purified oocysts supplemented with 100 mg oocyst-negative faecal material collected from a specific pathogen free chicken to mimic the absence of a flotation step. Briefly, to the pelleted oocysts an equal volume of autoclaved glass ballotini beads measuring ∼0.25–0.5 mm in diameter (Sigma–Aldrich, USA) were added and covered with a minimum volume ASL buffer (out of total 1.4 ml to be used for DNA isolation) supplied with the DNA extraction kit or sterile TE buffer. The oocysts were then disrupted by vortexing (India; Spinix Vortex Shaker, Tarsons, India; maximum speed) or beadbeating (Egypt, Libya and UK, Mini Beadbeater-8, Biospec Products, Bartlesville, USA; set to homogenise) for two minutes. Then, the remaining buffer ASL was added to the tube and thoroughly mixed. The suspension was then heated for 5 min at 70 °C and processed as per the QIAamp DNA Stool kit protocol. The DNA was eluted twice in 100 μl TE buffer as recommended by the manufacturer and quantified using absorbance at 260 and 280 nm.
4
Aflatoxin Detection Protocol
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Apparatus A mixer/grinder (Philips India Pvt. Ltd, Thane, India) was used for sample homogenization. A Spinix vortex shaker (Tarsons, Kolkata, India), high-speed centrifuge (Kubota Corp., Tokyo, Japan), and IAC kit (Aflaprep V R M 25) were used for the analysis.
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