26 June 2014 Extracting the DNA

Journal

Processing the Data

Once we return with samples from the field, remember we have those filters full of microbes that we collected from the water, we have to process the samples and isolate the DNA. If you want to read more about field sampling check out my I-Series Journal.

Cracking the Tubes

Sterivex tubes — You can see the differences in colors between some of the sterivex tubes. These contain populations of microbes collected from different field sites. These tubes are cracked open and then the DNA is extracted.

The first step in processing a sample is to remove the filter (along with all of the microbes) from the plastic container. We do this through a process called "cracking". We literally crack open the tubes with pliers and remove the filter paper. This filter paper is then placed in another tube and stored until we are ready for the extraction process.

Cracking the tubes — Here Byron Crump has removed the filter that is covered in microbes from the sterivex filter that was collected in the field. He cracked the tubes open and removed the paper and this paper is now saved and stored for further extraction.

DNA Extractions

Extracting the DNA is a process going through different steps and processes to create conditions that cause the cell to break open are release DNA. We are only interested in the DNA and not any of the other parts so we have to remove the extra junk.

Lauren preparing DNA — Lauren Watel adds enzymes to the filters containing microbes collected in the field to start the process of DNA extraction.

The first thing we do is add some different enzymes to help break up the cell membrane. Each cell is surrounded by a membrane, it is meant to help contain everything, we need to break open that container. So to do this, enzymes help us do some of the work. The membrane is made up of different components and these enzymes (which are just specialized proteins that make reactions happen faster) work to break it open.

Incubating the DNA — The DNA goes through a series of rapid freezing at -80ºC and being incubated in a hot water bath at 37ºC to shock the cells and help destroy their membranes.

We then also go through a process of freezing and rapidly thawing the cells to disrupt them and help break them open. We go from -80ºC in a freezer to 37ºC in the span of just a few seconds.

Decanting the DNA — After the tubes are spun in a centrifuge to separate the different layers by density we carefully remove and save the top layer that contains the DNA- it is shown here in the purple colored liquid that is going into the pipette.

Our next step involves mixing the cell parts with some different solutions to help separate the DNA from the other stuff. The DNA is much lighter than the other parts of the cell so they start to separate by weight.

Extracting the DNA — After ethanol is added to the tube, the DNA condenses and forms a small clump that is spun with the centrifuge to the bottom of the tube. We then carefully remove all remaining liquid with the pipette, so that just the small clump of DNA, which is located at the bottom of the tube, is left behind for storage and transfer to Oregon State University.

Eventually we end up with just the DNA in a little tiny clump in the tube! It's a long process, but careful work leads to easy success. These DNA clumps can then be frozen and sent back to Oregon for further analysis. I wrote a bit about how this analysis takes place when I visited Byron's lab back in May. That journal is found here.

We have to keep everything as sterile as possible to minimize introducing any unwanted bacteria from other environments. We also keep things carefully labeled and organized so that samples are not lost or mixed up.

Species Journal

Today was spent in the lab processing the samples from the I-Series day of sampling. It was cloudy and rained lightly off and on, so I didn't mind staying inside. Once again, I have no new species that I viewed to add to my species journal, but I hope you enjoy this one of a species that does actually live in the area.

A Reminder

Depending on when you are reading this, tomorrow, Friday, June 27th is my LIVE PolarConnect web event. Byron and I will be talking about life at Toolik and in the Arctic, and briefly explaining his research and findings thus far in the study. Tune in! Instructions for registration and access are found here

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