Flies were homogenized in 200 μl of cold PBS. The homogenate was heat-treated at 70 °C for 5 min to inactivate endogenous enzymes. Samples were centrifuged for 3 min at 16,100 × g and the supernatant collected. Protein concentration was measured using a Bradford assay (Bio-Rad 5000006). Samples were diluted with PBS and 90 μl of heat-treated homogenate were incubated with either 20 μl of amyloglucosidase (Sigma-Aldrich A7420) or 20 μl of PBS. To create a glycogen standard curve, 50 μl of glycogen standards (Ambion AM9510) were treated with either 50 μl of amyloglucosidase or 50 μl of PBS. All samples were incubated at 37 °C for 1 h. Then, 30 μl of each sample was added to a 96-well plate. Next, 100 μl of Infinity Glucose Hexokinase reagent (Thermo TR15421) was added to all samples and incubated at room temperature for 15 min. The absorbance of samples was then measured at 340 nm and normalized by subtracting the absorbance of the free glucose of untreated samples from the absorbance of the total amount of glucose present in samples treated with amyloglucosidase. Glycogen content was then calculated based on the normalized glycogen standard curve.
Am9510
Manufactured by Thermo Fisher Scientific
The AM9510 is a laboratory instrument designed for performing nucleic acid amplification tests. It is a thermal cycler capable of executing various polymerase chain reaction (PCR) protocols. The core function of the AM9510 is to precisely control the temperature and cycling conditions required for the amplification of DNA or RNA samples.
Automatically generated - may contain errors
Lab products found in correlation
Trizol reagent, by Thermo Fisher Scientific (2 mentions)
Infinity glucose hexokinase reagent, by Thermo Fisher Scientific (1 mentions)
Q700 sonicator, by Qsonica (1 mentions)
Proteinase inhibitor cocktail, by Merck Group (1 mentions)
Rna screentape on tapestation, by Agilent Technologies (1 mentions)
S7899, by Merck Group (1 mentions)
Dnase 1, by Roche (1 mentions)
Qubit rna hs assay kit, by Thermo Fisher Scientific (1 mentions)
5 protocols using am9510
1
Glycogen Quantification in Fly Homogenates
2
Chromatin Immunoprecipitation and Real-time PCR
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After treatments, cells were cross-linked with 1% (w/v) formaldehyde for 5 min at room temperature. Ice cold glycine (125mM) was applied to quench formaldehyde. Then the cells were washed twice with ice cold PBS and collected. Cross-linked cells were resuspended in 1 ml immunoprecipitation (IP) buffer (150mMNaCl, 50mM Tris-HCl (pH 7.5), 5mM EDTA, NP-40(0.5%), Triton X-100 (1%), and added cocktail proteinase inhibitor (Sigma). Cell lysate were sonicated for 10 x 30 s with 30 s break using Qsonica Q700 sonicator. Then the sonicated cells were centrifuged and the supernatant was performed for immunoprecipitation. Each antibody was incubated with lysate overnight with rotation at 4 °C. And then the lysate was incubated with pre-blocked protein G beads with rotation for 10h at 4 °C. Then the beads were rinsed with high salt IP buffer supplemented with 500mM NaCl, IP buffer and finally resuspended in TE buffer (pH 8.0). Then Proteinase K (Qiagen #1018832) was added in DNA-protein complex for digestion overnight at 65 °C. Finally the DNA was purified by phenol-chloroform extraction and ethanol precipitation with the presence of glycogen (Ambion #AM9510). The purified DNA was used for real-time PCR.
3
RNA Fragmentation for m6A-seq
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RNA fragmentation is based on the previously described m6A-seq protocol [27 (link)] with a few modifications: the total volume of approximately 3 to 5 μg total RNA was adjusted to 18 μl with RNase-free water. The amount of 2 μl of 10X RNA Fragmentation Buffer (100 mM Tris-HCl, 100 mM ZnCl2 in nuclease-free H2O) was added and incubated in a preheated thermal cycler for approximately 5 to 6 minutes at 70 °C. The reaction was stopped by adding 2 μl of 0.5 M EDTA. Then added to the mixture was 178 μl of H2O, 20 μl of sodium acetate (3 M [pH 5.2]; S7899; Sigma-Aldrich, St. Louis, MO), 14.4 μl of glycogen (5 mg/mL; AM9510; Thermo Fisher Scientific), and 500 μl of 100% ethanol, and the mixture was incubated at −80 °C overnight. Fragmented RNA was pelleted by centrifuge, washed once with 75% ethanol, and resuspended in ultrapure H2O (10 μl H2O per 1 μg human total RNA). The size distribution of fragmented RNA was assessed using High Sensitivity RNA Screentape on TapeStation (5067–5576; Agilent Technologies; Santa Clara, CA). The total RNA was chemically fragmented into approximately 200-nt-long fragments.
4
RNA Extraction Using TRIzol Reagent
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RNA was extracted with TRIzol reagent (15596010, Thermo Fisher Scientific). Briefly, snap-frozen cell pellets were suspended in 500 μL TRIzol, followed by a 5 min incubation at RT and addition of 0.1 mL chloroform. The samples were vortexed for 15 s, or until homogeneity in colour. After a 3 min incubation at RT, samples were centrifuged at 14,000 rpm for 15 min at 4°C to allow phase separation. The top aqueous RNA-containing layer was transferred to fresh tubes; 1 μL RNAse-free glycogen (AM9510, Thermo Fisher Scientific) and 250 μL isopropanol (I91516, Sigma) were added. Samples were mixed by inverting three to four times and incubated for 10 min at RT. A 30 min centrifugation at 14,000 rpm at 4°C was performed to pellet the RNA, which was subsequently washed with 250 μL of 75% ethanol. The supernatant was removed, and the pellet air-dried before suspending in 50 μL nuclease-free water. The samples were vortexed for 15 s and then incubated at 55°C for 10 min to completely dissolve the RNA. The samples were stored at −80°C.
5
RNA Extraction and Purification from Cultured Cells
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Total RNA from cells in culture was extracted using Trizol reagent (15596018; Thermo Fisher Scientific, Waltham, MA). Due to the presence of m6A in DNA [45 (link)], total RNA was treated with DNase I (04 716 728 001; Roche Diagnostics, Indianapolis, IN) for 20 minutes at 37 °C to remove DNA contamination. The RNA was precipitated using glycogen (25 μg/mL final) (5 mg/mL; AM9510; Thermo Fisher Scientific) and isopropanol at −30 °C for 2 hours. The precipitated RNA was then washed with 70% ethanol. The final pellet was resuspended in ultrapure H2O. The concentration of total RNA was measured by Qubit RNA HS Assay Kit (Q32855; Thermo Fisher Scientific).
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