Rotavirus genome extraction and visualization were performed as previously described (96 (link)). Briefly, MA104 cells at a density of 3 × 105 cells per well in a 6-well multiwell plate were infected with a virus at MOI of 10 VFU/cell and incubated in 1 mL serum-free DMEM until complete cytopathic effect was reached. The cells and supernatant were harvested, followed by three cycles of liquid nitrogen freeze and 37°C water bath. Then, the samples were mixed vigorously at a ratio of 1:1 with saturated phenol solution pH 4.3 (Merck) and centrifuged for 15 min at 13,000 rpm. The aqueous phase was recovered, and the previous step was repeated. Then, the RNA in the recovered aqueous phase was precipitated by mixing with 0.1 vol 3M Na Acetate at pH 5.2 and 2 vol of 100% ethanol. Samples were incubated for 30 min at −80°C and then centrifuged at 13,000 rpm for 30 min and 4°C. The pellet was resuspended in 15 μL distilled water and mixed with 10 μL Gel Loading dye 6X (New England BioLabs). The samples were migrated in an 8.5% SDS-polyacrylamide gel at 180 volts for 120 min, followed by staining with GelRed Acid gel Stain (Biotium) for 30 min. Images were acquired at Odyssey FC (LI-COR Biosciences).
Gel loading dye 6x
Manufactured by New England Biolabs
Gel Loading Dye 6X is a concentrated solution designed for use in gel electrophoresis. It increases the density of samples, allowing them to sink to the bottom of the sample wells. The dye also provides visual tracking of the electrophoresis progress.
Automatically generated - may contain errors
2 protocols using gel loading dye 6x
1
Rotavirus Genome Extraction and Visualization
2
Fungal DNA Extraction and ColonyPCR
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Genomic DNA was extracted from a small amount of mycelium collected from a PDA plate. It was put into a screw-cap microfuge tube together with 0.5-mm glass beads and disrupted using a Precellys 24 homogenizer. Three cycles at 5000 rpm were performed for 60 s with suspending for 5 s. Subsequently, 150 µL of 25 mM Tris-HCl buffer, pH 8.0 containing 50 mM glucose and 10 mM EDTA was added and the tube inverted 5 times. It was centrifuged for 1 min at 9500
x g and the supernatant transferred to another tube. For a cleaner sample, the centrifugation was repeated. Fivehundred µL of isopropanol was added to the clear supernatant and incubated at -20 °C for 2 h. The DNA precipitate was centrifuged at 9500 x g and 4 °C for 10 min and washed with 4 °C-cold 70% ethanol. The pellet was dried at 60 °C for 30 min and resuspended in 30 µL puri ed H 2 O.
ColonyPCR was performed with OneTaq DNA polymerase from NEB according to the manufacturer's manual. As forward primer, the sequencing primer (5pLHseq1, Table 4) that attaches in the promoter region was used. The reverse primer (3PcCDH_colPCR, Table 4) was designed to obtain a 300-bp fragment at the beginning of the gene. The PCR was analyzed by gel electrophoresis using 0.8% agarose gel, Gel Loading Dye (6X, NEB) and 2-Log DNA Ladder (NEB) in a horizontal Mini-Sub Cell electrophoresis system (Bio-Rad).
x g and the supernatant transferred to another tube. For a cleaner sample, the centrifugation was repeated. Fivehundred µL of isopropanol was added to the clear supernatant and incubated at -20 °C for 2 h. The DNA precipitate was centrifuged at 9500 x g and 4 °C for 10 min and washed with 4 °C-cold 70% ethanol. The pellet was dried at 60 °C for 30 min and resuspended in 30 µL puri ed H 2 O.
ColonyPCR was performed with OneTaq DNA polymerase from NEB according to the manufacturer's manual. As forward primer, the sequencing primer (5pLHseq1, Table 4) that attaches in the promoter region was used. The reverse primer (3PcCDH_colPCR, Table 4) was designed to obtain a 300-bp fragment at the beginning of the gene. The PCR was analyzed by gel electrophoresis using 0.8% agarose gel, Gel Loading Dye (6X, NEB) and 2-Log DNA Ladder (NEB) in a horizontal Mini-Sub Cell electrophoresis system (Bio-Rad).
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