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RAEiCKIwbKY0YHZhMFWg-DxcaWOZZSs0dC6lzDyGkKM4Y.step description "Add imaging buffer with desired ratios of Buffer C (500 mM), ethylene carbonate, and IS-CF660R at 1-2 nM final concentration. The exact concentration of IS may need to be adjusted depending on the target and based on the imaging kinetics." RAEiCKIwbKY0YHZhMFWg-DxcaWOZZSs0dC6lzDyGkKM4Y.assertion.
RAFt6stRZSIiuA-w5go4rzFRQJci2Ez9yYnRw60sQMcYQ.step description "DNA point accumulation for imaging in nanoscale topography (DNA-PAINT) facilitates multiplexing in superresolution microscopy but is practically limited by slow imaging speed. We have developed DNA-PAINT-ERS, where E=ethylene carbonate, R=repeating sequence, and S=spacer, for fast and multiplexed superresolution imaging with DNA-PAINT. Here we describe detailed procedures for DNA-PAINT-ERS including reagent preparation, sample labeling, as well as image acqusition and analysis." RAFt6stRZSIiuA-w5go4rzFRQJci2Ez9yYnRw60sQMcYQ.assertion.
step description "After incubating at room temperature for a few minutes, keep the solubilized nucleic acid in −20˚C for a short time storage or in −80˚C for a longtime storage. Figure 1 shows a schematic model of all the DNA and RNA isolation steps of this procedure." assertion.
step description "In this step, incubate samples at 65℃ for 10 min for lysing cells completely." assertion.
step description "Transfer the resulting solution to a sterile centrifuge tube (size=2 ml), and then mix sample by briefly vortexing until the sample is thoroughly resuspended." assertion.
step description "Discard the supernatant and wash the precipitate nucleic acid gently with 70% EtOH." assertion.
step description "Add 700 µl of cold isopropanol to precipitate RNA or DNA and then invert tubes 3-4 times to mix the solution." assertion.
step description "Dissolve the dry pellet in nuclease free water or TE buffer (Ph 8.0). Store DNA at 4 º celcius or -20 º celcius ." assertion.
step description "Grind mosquitoes in 1.5 ml eppendorf tube with micropistle in 50-100 µl 1X STE buffer ( 50mM Nacl,50mM Tris- HCL,100mM EDTA, Ph 8.0) along with 100mM sucrose. Add 1X STE buffer to a total volume of 300-500 µl for a single mosquito and 1 ml for mosquito pool like 4,6,8,10 numbers. Then add 1% SDS ,1% Triton -X , 10 µl/ ml Rnase A (20mg/ml), 20 µl/ ml Proteinase K (20mg/ml) and mix it." assertion.
step description "Wash preserved mosquitoes in sterile distilled water or phosphate buffer saline(PBS) to remove excess alcohol. Fresh mosquitoes can be ground directly." assertion.
step description "Keep the pellet at 37 º celcius for 10 minutes." assertion.
step description "Centrifuge at 12,000g for 10 minutes at 4º celcius .Transfer the supernatant to a fresh tube." assertion.
step description "Lyse for 1 hour 30 minutes at 37º celcius. Gently mix the tube by inverting every 15 minutes." assertion.
step description "Add 2 volumes of cold 95% ethanol and mix gently by inversion. The tubes are incubated at for 45 min in -20°C freezer. It should not be for more time as some remaining phenolics and resin may also precipitate with DNA." assertion.
step description "After RNAse A treatment, add one volume of chloroform: isoamyl alcohol again and mix by inversions for 5 min. The tube is centrifuged for 10 min at 5000 × g at 4°C. Transfer the clear supernatant into new 2ml tube and add half volume of 5 M NaCl to the sample and mix gently by inversions." assertion.
step description "Take one gram of frozen finely powdered leaf tissues in a new 50 mL Falcon tube and mixed with the pre-heated extraction buffer (10 ml) for one sample. Breifly, vertex for 30sec to ensure leaf material is fully mixed with buffer. Fine grind is a key to obtaining high DNA quantity with lesser artifacts of resin." assertion.
step description "Dissolve pellet in molecular biology grade water and analyze DNA by agarose gel electrophoresis and spectrophotometer for its purity and integrity." assertion.
step description "Add 10 ml TE buffer into the mortar and carefully bring mycobacteria in suspension. Transfer this suspension to a 50 ml polypropylene tube." assertion.
step description "Incubate mycobacterial suspension at 80 °C for 1 h in a water bath. This step will facilitate loosening of the mycobacterial cell wall and will also inactivate pathogenic mycobacteria." assertion.
step description "Harvest mycobacteria by centrifuging 20-40 ml log phase culture (O.D.600 ≈ 0.8) at 2,000 x g for 10 min, room temperature. Carefully discard culture medium and add 10 ml TE (at room temperature) buffer to pellet. Mix gently with a pipette. Note: If isolating DNA from pathogenic mycobacteria such as M. tuberculosis, step 1 and 2 must be performed in a BSL-3 facility." assertion.
step description "Aspirate liquid without disturbing pellet. Keep tube open to air dry pellet. Do not over dry pellet, as over dried DNA will be difficult to dissolve." assertion.
step description "Wash DNA pellet by adding 10 ml of 75% ethanol. Centrifuge at 12,000 x g for 10 min, 4 °C." assertion.
step description "Add 100 µl 3 M sodium acetate solution per ml of aqueous phase obtained in step 10. Mix gently by inverting tube." assertion.
step description "Repeat steps 6 to 8 for achieving a higher purity of DNA." assertion.
step description "Centrifuge suspension at 12,000 x g for 10 min, 4 °C. Carefully transfer upper aqueous phase to a new 50 ml tube." assertion.
step description "Add 1 ml of 10% SDS solution (final concentration, ~1%) and 20 µl of 10 mg/ml Proteinase K solution (final concentration 20 µg/ml). Incubate for 2 to 4 h at 60 °C in a hot water bath." assertion.
step description "Wash (centrifuge in 100 x g for 10 min) twice with 10 ml of sterile PBS or sterile Dulbecco's modified eagle medium. The approximate yield of cells from 4 ml of blood varies between 107-108." assertion.
step description "The cells in interphase need to be aspirated without delay. If the tubes are kept standing for more than 10 min, PBMCs from the interphase will get disturbed and start settling down." assertion.
step description "Aspirate the whitish buffy coat (about 1 ml) (PBMCs) formed in the interphase between histopaque and medium." assertion.
step description "Centrifuge the tubes (without any delay) for 30 min at 100 x g in 4 °C in a swing-out bucket. Fixed angle rotors also can be used but would require more caution when separating cells in interphase." assertion.
step description "Gently layer the blood on the top of Ficoll Histopaque using a 1 ml auto pipette. The layering should be done very slowly that blood and Ficoll Histopaque should stay as two different layers." assertion.
step description "Cells can be treated with different antigens for different period of times and the supernatants can be analysed for cytokine levels. The cells can be analysed for phenotypic change, apoptosis or proliferation. Note: PBMCs are primary cells and cannot be cultured for more than one passage under normal conditions. Lymphocytes of PBMCs can be made to proliferate in vitro by mitogens e.g., Phytohaemagglutin or Concanavin-A etc over a period to 72-96 h. Monocytes generally are end cells and do not proliferate. In absence of mitogens the proliferation of PBMCs will be negligible." assertion.
step description "Discard medium and re-suspend the cell pellet in 1 ml of sterile Dulbecco's modified eagle medium." assertion.
step description "Wash cells (centrifuge at 100 x g for 10 min) with 10 ml of sterile DMEM (without FBS) twice. Note: Cold DMEM is not used routinely for washing lymphocytes from culture cavities while setting up cultures. Rather when monocytes bound tightly to plastic cavities are needed to be harvested pre-chilled DMEM can be used. " assertion.
step description "Collect about 4 ml of human venous blood sample in heparinised vials and mix well by gently inverting the tubes several times." assertion.
step description "Discard the supernatant and wash the precipitate nucleic acid gently with 70% EtOH." assertion.
step description "Centrifuge tubes at 13700 g at 4℃ for 10 min. The white pellet will be visible on the bottom of the tubes.CRITICAL STEP Do not disturb bottom phases of the solution when you pipet the upper phase." assertion.
step description "Transfer the upper aqueous phase to a new tube (size= 1.5 ml)." assertion.
step description "Centrifuge at 13700 g at 4℃ for 10 min.CRITICAL STEP The PH of the extraction buffer is about 8-9, resulting in DNA and RNA precipitation, however, it could result in lower DNA and higher RNA concentrations in case of reducing the PH to around 6-7." assertion.
step description "Transfer the resulting solution to a sterile centrifuge tube (size=2 ml), and then mix sample by briefly vortexing until the sample is thoroughly resuspended." assertion.
step description "The RNA pellets are air-dried and dissolved in 100 µl RNase-free H2O by incubating at 65 °C for 15 min." assertion.
step description "RNA pellet was washed with 1 ml 70% RNase-free ethanol once and spin down by 7,500 x g for 5 min." assertion.
step description "RNA was precipitated by centrifugation at 12,000 x g for 10 min at 4 °C." assertion.
step description "The upper aqueous phase was transfer into a new tube and mixed with 1:1 (volume/volume) of 100% isopropanol, and then incubated for 10 min at room temperature." assertion.
step description "At the indicated time points (0, 2, 4, 8, 12, 16, 20, 24, 28 h post-infection), cells were rinsed with 10 ml Phosphate Buffered Saline (PBS) buffer once and lysed in 1 ml TRIzol for 5 min at room temperature." assertion.
step description "Ground samples (leaf, shoot, root, and recalcitrant samples, approximately 0.5-1 g) using 1 ml of the extraction buffer with or without liquid nitrogen in mortar and pestle that are sterilized.CRITICAL STEP The procedure are carried out at room temperature except the centrifugation steps (at 4℃) as well as the time of precipitating of the nucleic acid using the isopropanol (on ice).CRITICAL STEP Severely disrupt the tissues to create the glaze mode of samples" assertion.
step description "Discard the supernatant and wash the precipitate nucleic acid gently with 70% EtOH." assertion.
step description "Ground samples (leaf, shoot, root, and recalcitrant samples, approximately 0.5-1 g) using 1 ml of the extraction buffer with or without liquid nitrogen in mortar and pestle that are sterilized.CRITICAL STEP The procedure are carried out at room temperature except the centrifugation steps (at 4℃) as well as the time of precipitating of the nucleic acid using the isopropanol (on ice).CRITICAL STEP Severely disrupt the tissues to create the glaze mode of samples" assertion.
step description "Add 700 µl of cold isopropanol to precipitate RNA or DNA and then invert tubes 3-4 times to mix the solution" assertion.
step description "Centrifuge at 13700 g at 4℃ for 10 min.CRITICAL STEP The PH of the extraction buffer is about 8-9, resulting in DNA and RNA precipitation, however, it could result in lower DNA and higher RNA concentrations in case of reducing the PH to around 6-7" assertion.
step description "Dissolve the dry pellet in nuclease free water or TE buffer (Ph 8.0). Store DNA at 4 º celcius or -20 º celcius" assertion.
step description "Wash preserved mosquitoes in sterile distilled water or phosphate buffer saline(PBS) to remove excess alcohol. Fresh mosquitoes can be ground directly." assertion.
step description "Wash preserved mosquitoes in sterile distilled water or phosphate buffer saline(PBS) to remove excess alcohol. Fresh mosquitoes can be ground directly." assertion.
step description "Keep the pellet at 37 º celcius for 10 minutes" assertion.
step description "Wash the pellet with 70% ethanol" assertion.
step description "Centrifuge at 12,000g for 30 minutes at 4º celcius and then remove the supernatant" assertion.
step description "Transfer the very clear supernatant to a fresh tube , add two fold volume cold isopropanol and keep it for 1 hour at -20º celcius" assertion.
step description "Centrifuge at 12,000g for 10 minutes at 4º celcius .Transfer the supernatant to a fresh tube" assertion.
step description "After incubation, the tubes were centrifuged at 5000 × g for 10 min at 4°C and the supernatant was gently removed. The pellet is washed two times with 1ml of 70% ethanol and the DNA is pellet by 5000 × g at 4°C for only 5 min. The supernatant is discarded and the pellet is air-dried (10 min). The pellet are allowed to re-suspend in 50 μL of TE (10 mM Tris. HCl pH 8.0; 1 mM EDTA pH 8.0)." assertion.
step description "After RNAse A treatment, add one volume of chloroform: isoamyl alcohol again and mix by inversions for 5 min. The tube is centrifuged for 10 min at 5000 × g at 4°C. Transfer the clear supernatant into new 2ml tube and add half volume of 5 M NaCl to the sample and mix gently by inversions" assertion.
step description "Transfer 1ml of supernatant to each 2ml Eppendorf tubes already containing 1 ml of chloroform: isoamyl alcohol (24:1). Mix supernatant and chloroform: isoamyl alcohol by gentle inversions for 10 min and subsequently place the tube on ice for 10 min." assertion.
step description " The falcon tube was kept into the 65°C incubator or water bath and mix gently by inversion after every 10 min till 45 min. After incubation, place the tube at room temperature for five min to reach to room temperature environment. Centrifuge the 50ml falcon tube for 5 min at 3000×g on room temperature. For next generation sequencing, the greater the genome size, lower is speed of initial centrifugation" assertion.
step description "If in step one the sample is not grinded with mortar and pestle then vortex the falcon tube for 5 min otherwise proceed to step 3" assertion.
step description "Dissolve pellet in molecular biology grade water and analyze DNA by agarose gel electrophoresis and spectrophotometer for its purity and integrity" assertion.
step description "Snap freeze pellet in liquid N2 and immediately transfer to a mortar. Using a pestle, crush bacteria while repeatedly adding liquid N2 (~20 ml) to it." assertion.
step description "Incubate mycobacterial suspension at 80 °C for 1 h in a water bath. This step will facilitate loosening of the mycobacterial cell wall and will also inactivate pathogenic mycobacteria." assertion.
step description "Aspirate liquid without disturbing pellet. Keep tube open to air dry pellet. Do not over dry pellet, as over dried DNA will be difficult to dissolve" assertion.
step description "Wash DNA pellet by adding 10 ml of 75% ethanol. Centrifuge at 12,000 x g for 10 min, 4 °C" assertion.
step description "Centrifuge tube at 12,000 x g for 10 min, 4 °C. Discard liquid carefully" assertion.
step description "Add 100 µl 3 M sodium acetate solution per ml of aqueous phase obtained in step 10. Mix gently by inverting tube" assertion.
step description "Add an equal volume of PCI mixture to the tube and mix contents by gently inverting tube for 5-10 min" assertion.
step description "Centrifuge at 12,000g for 30 minutes at 4º celcius and then remove the supernatant ." assertion.
step description "Transfer the very clear supernatant to a fresh tube , add two fold volume cold isopropanol and keep it for 1 hour at -20º celcius ." assertion.
step description "Add equal volume of phenol:chloroform(1:1) ,shake the tube well for 5 minutes and centrifuge at 12,000g for 10 minutes at 4º celcius ." assertion.
step description "Add 2 volumes of cold 95% ethanol and mix gently by inversion. The tubes are incubated at for 45 min in -20°C freezer. It should not be for more time as some remaining phenolics and resin may also precipitate with DNA." assertion.
step description "The falcon tube was kept into the 65°C incubator or water bath and mix gently by inversion after every 10 min till 45 min. After incubation, place the tube at room temperature for five min to reach to room temperature environment. Centrifuge the 50ml falcon tube for 5 min at 3000×g on room temperature. For next generation sequencing, the greater the genome size, lower is speed of initial centrifugation" assertion.
step description "If in step one the sample is not grinded with mortar and pestle then vortex the falcon tube for 5 min otherwise proceed to step 3." assertion.
step description "The IBV-infected cells were incubated at 37 °C in 5% CO2." assertion.
step description "RNA concentration and purity were determined by NanoDrop." assertion.
step description "The RNA pellets are air-dried and dissolved in 100 µl RNase-free H2O by incubating at 65 °C for 15 min." assertion.
step description "Transfer the very clear supernatant to a fresh tube , add two fold volume cold isopropanol and keep it for 1 hour at -20º celcius" assertion.
step description "Grind mosquitoes in 1.5 ml eppendorf tube with micropistle in 50-100 µl 1X STE buffer ( 50mM Nacl,50mM Tris- HCL,100mM EDTA, Ph 8.0) along with 100mM sucrose. Add 1X STE buffer to a total volume of 300-500 µl for a single mosquito and 1 ml for mosquito pool like 4,6,8,10 numbers. Then add 1% SDS ,1% Triton -X , 10 µl/ ml Rnase A (20mg/ml), 20 µl/ ml Proteinase K (20mg/ml) and mix it." assertion.
step description "Wash preserved mosquitoes in sterile distilled water or phosphate buffer saline(PBS) to remove excess alcohol. Fresh mosquitoes can be ground directly." assertion.
step description "Wash the pellet with 70% ethanol ." assertion.
step description "Keep the pellet at 37 º celcius for 10 minutes." assertion.
step description "Dissolve the dry pellet in nuclease free water or TE buffer (Ph 8.0). Store DNA at 4 º celcius or -20 º celcius ." assertion.
step description "Centrifuge the tube for 10 min at 5000 × g at 4°C. Transfer the upper aqueous phase into new 2ml tubes and add 5μl RNAse A (10 mg/mL). Place the tube for 30 min at 37°C." assertion.
step description "After RNAse A treatment, add one volume of chloroform: isoamyl alcohol again and mix by inversions for 5 min. The tube is centrifuged for 10 min at 5000 × g at 4°C. Transfer the clear supernatant into new 2ml tube and add half volume of 5 M NaCl to the sample and mix gently by inversions." assertion.
step description "After RNAse A treatment, add one volume of chloroform: isoamyl alcohol again and mix by inversions for 5 min. The tube is centrifuged for 10 min at 5000 × g at 4°C. Transfer the clear supernatant into new 2ml tube and add half volume of 5 M NaCl to the sample and mix gently by inversions" assertion.
step description "After incubation, the tubes were centrifuged at 5000 × g for 10 min at 4°C and the supernatant was gently removed. The pellet is washed two times with 1ml of 70% ethanol and the DNA is pellet by 5000 × g at 4°C for only 5 min. The supernatant is discarded and the pellet is air-dried (10 min). The pellet are allowed to re-suspend in 50 μL of TE (10 mM Tris. HCl pH 8.0; 1 mM EDTA pH 8.0)." assertion.
step description "The IBV-infected cells were incubated at 37 °C in 5% CO2" assertion.
step description "Cell lysates were transfer into eppendorf tubes and one-fifth (volume/volume) of chloroform was added to each tube." assertion.
step description "The upper aqueous phase was transfer into a new tube and mixed with 1:1 (volume/volume) of 100% isopropanol, and then incubated for 10 min at room temperature." assertion.
step description "RNA was precipitated by centrifugation at 12,000 x g for 10 min at 4 °C." assertion.
step description "Cells were seeded in 100-mm-diameter dishes and infected with either 2 PFU of live IBV per cell or the same amount of UV-inactivated IBV (UV-IBV) at 37 °C. Excess virus in the medium was removed by replacing with fresh medium at 1 h post-infection." assertion.
step description "RNA was precipitated by centrifugation at 12,000 x g for 10 min at 4 °C." assertion.
step description "RNA pellet was washed with 1 ml 70% RNase-free ethanol once and spin down by 7,500 x g for 5 min." assertion.
step description "The RNA pellets are air-dried and dissolved in 100 µl RNase-free H2O by incubating at 65 °C for 15 min." assertion.
step description "RNA pellet was washed with 1 ml 70% RNase-free ethanol once and spin down by 7,500 x g for 5 min." assertion.
step description "At the indicated time points (0, 2, 4, 8, 12, 16, 20, 24, 28 h post-infection), cells were rinsed with 10 ml Phosphate Buffered Saline (PBS) buffer once and lysed in 1 ml TRIzol for 5 min at room temperature." assertion.