Last week the final pieces of equipment for Barcode the Lake arrived; a thermalcycler, micropipette and a gel electrophoresis box.
Micropipettes are the monkey wrench of modern biology. The tools allow users to move exact amounts of liquids with high levels of accuracy. The pipette we have can precisely move 2 to 20 µl (microliters). Approximately 60 µl make up a drop of water, so this device allows the user to move about 1/30th of a drop of water with accuracy of +/- 0.05%! We will use the micropipette to move DNA, enzymes, and other biological materials. Here is a video on how to use a micropipette: https://youtu.be/p-OPOYbeZP0
Thermalcylcers are instruments that can quickly change temperatures allowing biologists to harness the power of different types of enzymes. We will be using our thermalcylcer to harness the power of DNA polymerases to make copies of DNA segments whose sequence can help us determine the identity of various organisms. The technique we will deploy is called the Polymerase Chain Reaction or PCR for short. The process was developed in Emeryville, California in the early 1980s by Kary Mullis at Cetus Corporation. The technique was a big step in modern biology as it allowed scientists to quickly and easily amplify small sections of DNA from entire genomes with high precision. The process of isolating enough DNA to do further molecular biology work with use to take months, but after the invention of PCR and subsequent development it now only takes a few hours. Here is a great animation that explains how the PCR process works: http://learn.genetics.utah.edu/content/labs/pcr/
Gel electrophoresis is a technique that is used to separate molecules based upon their size and charge. The box is a simple circuit with gel that sits between a positive and negative terminal that molecules can be loaded in. Positive charged molecules will move through the gel towards the negative terminal and negatively charged molecules will move through the gel towards the positive terminal. We will employ gel electrophoresis as a quality control check to make sure that we have used our thermalcycler to amplify our DNA segments of interest. DNA molecules all have the same net negative charge to them so in a gel electrophoresis box they will separate just based upon their size and we will use a stain to visualize the DNA since its normal invisible to our eyes. The DNA segments we are targeting should be between 400 and 800 basepairs and we will want to confirm them before sending them off for sequencing. Here is a animation that helps explain how DNA gel electrophoresis works: http://learn.genetics.utah.edu/content/labs/gel/
Validation Run of Equipment
The equipment came with reagents to make sure that everything worked. The validation run was made with Lambda DNA ( a virus that attacks bacteria) and three sets of primers that all amplify different locations within the Lambda DNA. The thermalcycler run took 15 minutes, which is amazingly fast since normal PCR runs take 3 hours, but the system we've invested in uses some tricks to speed things up. The running of the gel took another 20 minutes.
Now that we know all of our equipment works we can move onto our fist validation run of a real sample to make sure our entire process works! Here is the state of the collection at this time and where that first sample will come from:
After the full process validation run and confirming a location to do our lab work in (wish we could have used the Rotary Nature Center) we can start working with our first cohort of Barcode the Lake community scientists!!