DNA Isolation - C.gigas Larvae from Emma OA Experiments

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: