Isolated gDNA using DNazol from the following larval samples for potential MBD selection and bisulfite sequencing:
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Added 500uL of DNAzol to each tube, transferred to 1.5mL tube and homogenized with disposable pestles
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Added additional 500uL DNAzol to each homogenized sample and mixed by inversion.
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Incubated 10mins at RT
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Pelleted debris by spinning 10,000g, 10mins, @ RT
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Transferred supes to new tubes
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Added 500uL of 100% EtOH to each; mixed by inversion; incubated 10mins @ RT
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Pelleted DNA by spinning 5,000g, 4mins, @ RT
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Discarded supes
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Washed DNA with 1mL 70% DNAzol/30% EtOH solution
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Spun 1000g, 1min, @ RT
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Discard supes
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Washed DNA with 1mL 75% EtOH
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Spun 1000g, 1min, @ RT
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Discarded supes
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Spun 1000g, 1min, @ RT
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Removed residual EtOH with pipette; air dried samples for 5mins @ RT
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Added 20uL of Trish-HCl (pH = 8.0) to each sample; incubated 10mins @ RT; flicked tubes to help dissolve
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Spun 12,000g, 10mins, @ RT
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Transferred supes to new tubes
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Spec’d on NanoDrop 1000 (ThermoFisher) and recovered solution from each sample due to limited sample volume
Results:
