Protocol for the extraction of genomic DNA for use in PCR and Next Generation Sequencing. Yeilds total DNA that has been proteinase and RNase treated and is ready for downstream applications.

#DNeasy Blood and Tissue Kit (DNeasy Blood and Tissue Kit, Qiagen, Catalog # 69504)

• DNeasy Kit Manufacturer’s Instructions
• “For purification of total DNA from animal blood and tissues and from cells, yeast, bacteria, or viruses”
• All centrifugation steps are carried out at room temperature (15–25°C) in a microcentrifuge.
• Vortexing should be performed by pulse-vortexing for 5–10sec.
• Add ethanol to buffers once initially as stated in manufacturer’s instructions

Protocol for Isolation of total DNA from Animal Tissues (Spin-Column Protocol)

Procedure

• Add 0.180ml of Buffer ATL* to no more than 25mg of tissue in sterile RNAse DNAse free microfuge tube
• Homogenize tissue and buffer mix with teflon microfuge pestle
• Add 20µl of Proteinase K (included in kit) and mix by vortexing
• Incubate at 56°C until tissue is lysed (1-3hr)
• Add 4µl of RNase A (100mg/ml, not included in kit) and mix by vortexing
• Incubate at room temperature for 2min
• Vortex for 15sec
• Add 0.2ml Buffer AL to sample and mix by vortexing
• Add 0.2ml ethanol (96-100%) to sample and mix by vortexing
• Add the mixture to the DNeasy Mini spin column in collection tube
• Centrifuge at 6,000g for 1min and discard flow through and collection tube
• Place spin column in new collection tube
• Add 0.5ml Buffer AW1
• Centrifuge at 6,000g for 1min and discard flow through and collection tube
• Place spin column in new collection tube
• Add 0.5ml Buffer AW2
• Centrifuge at 20,000g for 3min and discard flow through and collection tube

Dry column completely, Do not allow column to come in contact with flow through when moving to new tube

• Place spin column in new 1.5ml or 2ml microfuge tube (not included in kit)
• Add 0.2ml of Buffer AE directly to the membrane
• Incubate at room temperature for 1min
• Centrifuge at 6,000g for 1min
• Aliquot DNA into 4 tubes (3µl for gel, 3µl for Qubit, 2x ~90µl for downstream applications

For maximum DNA yeild repeat elution

• Place spin column in new 1.5ml or 2ml microfuge tube (not included in kit)
• Add 0.1ml of Buffer AE directly to the membrane
• Incubate at room temperature for 1min
• Centrifuge at 6,000g for 1min
• Discard spin column
• Retain single tube of DNA as backup