I am now entering the analysis phase of my project where I process all of the blood samples I've taken and figure out if the birds are harboring any parasites. Here's a breakdown of the analysis steps that I've undertaken so far:
Collection
When blood is initially drawn from the bird in the field it is placed into a tube filled with lysis buffer. This serves to stabilize the blood at room temperature and also to lyse (break open) the red blood cells so that the DNA can be accessed. Back in the lab the tubes are put into a -80°C freezer for storage.
Extraction
Next comes the extraction phase where the DNA is separated out from the blood sample. Blood is full of many other materials including cell membranes, proteins, serum, etc. but what we're really interested in is the parasite DNA (if it's there.)
Extraction consists of a digestion phase and a purification phase. Digestion serves to further break down the cells beyond just lysis and then purification separates out the DNA from all the extra "stuff" in the mixture.
In Digestion we take the frozen blood sample and add it to a tube filled with digestion solution.
Then it gets incubated at 55°C overnight.
Allison models the incubator while I reflect.
Purification consists of several stages of washing and centrifuging to separate DNA out of the solution. In the end we have an aqueous (in water) sample of DNA, called an elution, that is then stored at -40°C until it's next needed.
PCR
PCR stands for polymerase chain reaction. The process involves targeting a specific segment of DNA and making many copies of it so we can have a workable quantity for study. It was such a significant contribution to science that the inventors were awarded the 1993 Nobel Prize in Chemistry.
In my case the segment targeted is one unique to blood parasites. Each of us in the lab has their own PCR kit consisting of all materials needed.
A PCR master mix is made up and pipetted out into individual tubes. Then the elutions from the Extraction step are added.
The tubes are then put into the thermocycler which can be programmed with specific cycles of hot and cool to unwind and copy the DNA segments.
The tubes are then put into the thermocycler which can be programmed with specific cycles of hot and cool to unwind and copy the DNA segments.
This process usually takes several hours. The end result is a PCR product that can be run through a gel.
Gel Electrophoresis
Gel electrophoresis is essentially the "read out" step following PCR. It tells me whether or not I have an infected bird or not.
Making up an agarose gel is likely something you did in high school biology. Agarose is dissolved in 1xTBE and distilled water then poured into a mold to set.
After the gel is set the PCR product is pipetted into the wells along with controls to compare my samples to known outcomes. A ladder with different lengths of DNA segments is also loaded to show DNA is properly moving through the gel. In the picture below the ladder is blue. I've loaded my positive control on both tiers right next to the ladder.
Making up an agarose gel is likely something you did in high school biology. Agarose is dissolved in 1xTBE and distilled water then poured into a mold to set.
I'd like to point out that our very high tech gel cooker is a microwave.
After the gel is set the PCR product is pipetted into the wells along with controls to compare my samples to known outcomes. A ladder with different lengths of DNA segments is also loaded to show DNA is properly moving through the gel. In the picture below the ladder is blue. I've loaded my positive control on both tiers right next to the ladder.
Then the gel is placed into a liquid bath and hooked up to a gel electrophoresis machine. DNA is negatively charged so when a voltage is applied the segments are attracted to the positive charge and travel through the gel.
The rule for hooking up the wires with the correct charge is "Run towards Red and Black goes in Back." You can adjust the time and voltage you run your gel with to control how far your samples travel.
Next the gel is stained and washed then placed in an imaging machine.
Here's what it looks like when it's all said and done. This is the printout that I tape in my lab book:
I tested 18 of my samples here. Two of them are positive and the rest are negative. This is both good and bad. Good that the birds are uninfected but bad because I need at least some of them to be infected so I can compare their songs.
I have to say that even though lab work is less frustrating than it was initially I still prefer field work. Nothing beats being outdoors and working with the species you're working to conserve.
