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