Genomic DNA from fresh or frozen tissue

Overview

The protocol was adapted from the Puregene Genomic DNA purification kit by Gentra. Generally start with 50 to 100 mg fresh or frozen tissue (about the size of a pencil eraser) and expect to get between 20 to 150 µg DNA. If there is more tissue, or it is possible to scale up and use a dounce homogenizer in the first step instead of using a razor blade to mince the tissue.

Protocol

• Mince tissue using a clean razor blade. Do this on a clean sheet of weighing paper. Throw away paper and clean razor in between samples.
• Place the tissue in a 1.5 ml eppe and add 1 ml cell lysis buffer containing 5 µl of 20 mg/ml Proteinase K solution.
• Vortex sample extensively
• Incubate in 55°C shaker at 250 rpms for at least 4 hrs, preferably overnight.

Cell lysis buffer 100 ml

• Don't forget to add the Proteinase K just before incubating overnight
• Add 1 µl of 10 mg/ml RNase A solution and invert tube 25 times
• Incubate 37°C for 30 minutes.
• Cool to room temp by placing on ice for 3 minutes
• Add 333 µl Protein Precipitation solution and vortex vigorously for 30 seconds

Protein Precipitation solution 50 ml

• Cool to room temp by placing on ice for 3 minutes
• Centrifuge 14,000 rpm 10 minutes. Proteins should pellet. If pellet is weak, vortex again and incubate on ice for 5 minutes, then recentrifuge.
• Pour supernatant with DNA into a clean 15 ml centrifuge tube
• Add equal volume (~1 ml) 100% isopropanol and mix by gently inverting 50 times
• Centrifuge 3500 rpm (2000g) for 3 minutes.
• Pour off supernatant
• Add 1 ml 70% ethanol and invert tube several times to wash the pellet
• Carefully transfer the washed pellet along with the ethanol to a 1.5 ml eppe tube.
• Centrifuge 14,000 rpm for 5 minutes (ideally at 4°C)
• Carefully pour off ethanol. Invert tube and air dry
• Resuspend DNA pellet in 50 to 500 µl TE depending upon size of pellet
• Optional incubation at 37°C to resuspend DNA
• Store at 4°C, or -20°C for long term.