Getting DNA from tissue samples

If our project is funded, our first step in the library construction process will be to extract DNA from the peacock bass samples we have. As I have been extracting DNA for another project I'm working on (on king mackerel population genetics), I thought I'd show you what that looks like.

The DNA extraction/isolation kits that we use process up to 10 milligrams of tissue at a time, so the first step is to subsample our tissues to get approximately that much of each sample. I extract DNA in batches of 88 samples to save time, but it does require more organization and care not to cross contaminate samples. I cut each sample on its own square of aluminum foil and heat my forceps with ethanol and a lighter between each sample.

 

After each sample is cut and placed in the deep well plate, we add a lysing agent and an enzyme that digests proteins and place the plate on a rocking heat plate. The heat helps the chemicals and enzyme to decompose the cells, spilling the DNA that was sequestered away in the cell's nucleus, into solution. 

 

However, there are lots of agents that were also in the cell that will degrade the DNA now that it's all mixed together, so we have to separate the DNA from all the other components of the cells. The kit we use, the Omega Mag-bind kit, uses special metal beads and an apparatus with rare earth magnets to separate the DNA. First, the tissue digestion is mixed with the beads and ethanol. The ethanol helps the DNA to adhere onto the beads, which are mixed thoroughly into the solution. The beads are a rust-colored brown.

 

Next, the plate is placed onto the magnet apparatus. This is essentially a small platform with magnetic rods that stick up between the wells. The beads are drawn towards the rods and stick against the sides of the wells. We can then use a pipette to draw off the liquid in the wells, which contains the cell components that we don't want mixed with the DNA.

 

The magnet is removed and the beads washed several times to remove all of the remaining cell components, and finally the beads are mixed with a light water-based buffer solution into which the DNA dissolves. Once more on the magnets and we remove the DNA elutions to further process the DNA for library construction. With any luck, we will recover between 1 to 5 micrograms of DNA from each sample to use in the DNA restrictions and ligations. More about that in a future lab note.