Extracting DNA might sound like something that you would only see in a Marvel movie for the creation of some amazing, all-powerful Captain Universe. However, even you can do this in the comfort of your own lab! All it takes is some fancy equipment, potentially dangerous chemicals, and some time and well-balanced centrifuging.
Let’s get started. Today, we’re going to be extracting DNA for fusarium PCRs.
Sounds unexciting? Think again. Just look at this beautiful rainbow of potato dry rot samples!
The first thing that we have to do to extract our fusarium DNA is to prepare a tube for it. Into this tube we need to place tiny glass beads to mechanically grind the cell walls and break up the cell tissue.
Now we need to transfer our DNA-containing material into our glass-bead filled tube. A sterilized toothpick works nicely for this job.
In it goes!
Time to bring in the chemicals. A dose of breakage buffer is put into each tube. The purpose of this buffer is to cause the cell to lyse, meaning that it busts open the cell membrane. Consequently, all of the cellular contents are released and merge together, forming a slurry of sloppy, goopy cell components and denatured proteins.
Next, we vortex these bad boys. This is where the beads actually do their mechanical cell-wall-breaking action.
Dangerous chemical #1 alert! Phenol:chloroform:isoamyl is added to separate the mixture into two phases: organic and aqueous. Phenol can cause very serious burns, so it’s important to exercise caution when working with it.
Wear your gloves! Put on those safety glasses! Do your work underneath the hood!
After the phenol has been safely added, a heavy duty centrifuge is put to use. This will spin the tubes around extremely fast and separate the contents into the two phases.
Eight thousand rotations per minute.
It’s like a super-charged tea cup ride.
Our two phases are now apparent and are separated by the interphase. The DNA has stayed in the aqueous phase (the clear liquid on top) and the other stuff (cellular components, proteins) have traveled to the bottom. The aqueous phase can be pipetted into another tube for the next step.
Dangerous chemical alert #2! Chloroform:isomyl is added and the tube is centrifuged, leaving another layer of clear DNA-containing phase on top that is pipetted into another tube. The purpose of the chloroform is to clean up any of the residual phenols. Chloroform has carcinogenic effects and presents another good opportunity to put those lab safety skills to use.
Sodium acetate is then added, which helps to precipitate the DNA.
Ethanol is the last thing added. DNA is soluble in water, but is insoluble in ethanol; adding ethanol makes the DNA fall out of solution.
Trudging onward! We lug out our trusty centrifuge to use again, this time spinning our solutions at a steady fifteen thousand rotations per minute. This step causes the DNA to gather at the bottom of the tube.
Now we gently pour off most of the remaining liquid, being careful not to dump the DNA pellet at the bottom (which isn’t very visible at this point) into the beaker.
Back into the centrifuge it goes! The rest of the liquid is poured off and a small globule of DNA is the only thing remaining, with some proteins attached. The proteins are what make the DNA visible and colored.
The DNA pellets are then left to dry.
We’re getting close to our final product. The end is in sight!
DNA re-suspension time!
A small amount of Tris-EDTA buffer is added to tube, which makes it easier for the DNA to dissolve. This buffer also contains RNase, which violently chops up any RNA that might have mistakenly found itself welcome in our final DNA product.
A final, twenty-minute rest in a mini dry bath re-suspended our DNA.
Finally, our finished product! It might look unimpressive but just think of all of the genetic information it contains wrapped up in a mere thirty microliters (thirty millionths of a liter).
What do we use the final products for? Fusarium PCRs!