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This very exciting item was donated by Dr. Matthew Breen for our 2011 Willem Wijnberg Cancer Fundraiser!

Spend a day working with the Breen Lab at NCSU


The Lucky Winner was

Wendy Djang


Wendy's Report of her visit.








As part of a world wide effort from Bernese Mountain Dog lovers, I participated in a fund raiser to generate essential funds to be awarded to Dr Breen and his research program. I was chosen at random from a considerable and generous pool of donors to win a day in the lab!

It took me a bit to find the perfect date to visit with Dr Breen and his staff at the NCSU Canine Genomics lab, but on December 15th last year my husband Bill and I learned how to isolate DNA from blood samples we had taken from our dogs a few days before.

I brought Churchill and his Golden Retriever siblings Riley and Mia to the lab to have blood drawn ahead of our visit. It was quite a change of pace for the dedicated research building to have three hairy beasts show up and immediately hog all the attention from students and staff alike. I couldn’t have asked for a better group of dogs and we tried our best not to leave too many hair balls behind. I had the feeling no one really minded since they don’t get the chance to meet the dogs they are helping very often!

So with parking permits, agendas, protocols and lab coats in hand I returned with Bill a few days later to get started on our project. The work seemed daunting at first. We had a full day ahead of us and at least half the terms on our protocol were new to me. I had never held a pipette in my life and I think the last time I was in any sort of lab had to be high school. It didn’t matter though because we were patiently guided through everything from lab safety to vocabulary and instruments by the expert staff of researchers. I was learning something new and exciting being around this talented group of people.





To get started we became familiar with the lab. We had our samples, blood from each of the dogs, that needed to be used at room temperature and like a good chef we prepared for each stage by making sure kits, pipettors, centrifuge tubes, buffers, and QIAamp Midi columns were all prepared to go at each stage. The task of isolating DNA from a blood sample is always seen as magic on modern TV shows. It’s a squirt of this and a scrape of that, and just like that you’ve caught the criminal! Our protocol had at least 15 different steps and each step had several components that would need to be repeated for each of the Goldens and twice for Churchill so that we always had a balanced and even distribution of four in the centrifuge.






Our original starting point was just 2 ml of blood. We were measuring things with the pipettors from 2.4 ml and smaller. The details included things like measuring temperature, disposing of pipette tips for every single action, vigorous shaking, (YES! By hand! The sample mixed with buffer needed to yield a homogenous solution.)





Precise incubation times, more vigorous shaking. We had to ,make sure all liquid was spun to the bottom of the tubes so that no liquid remained at the cap. We then divided the solution and buffer again and then taking that which remained and placing it on to a QIAamp Midi column (a kind of specialized DNA filtration device used for each sample) followed by more incubation and more time spinning in the centrifuge.







It’s not a pretty color any more at this stage, but before you know it the result will be a crystal clear liquid in a small precious amount that will be tested by the spectrophotometer for.concentration and quality.





This was the stage where we learned whether our hard work during the day was precise enough to move forward. We were lucky to have so much guidance and patience from the research team. Our result was that we had beautiful samples, all four samples showing a predictable curve, indicating great yield and concentration.





We next had to determine if our techniques had resulted in high molecular weight genomic DNA, or if we had damaged it during isolation. For this we used agarose gel electrophoresis. For this we used the tiniest of pipette tips and the steadiest of hands we loaded our gel with the samples – just 10 microlitres (1/100 of 1 ml – yes, a fraction of one drop).





There are no fancy words needed to describe the medium that we were using. “Gel” is just right. Think, “JELLO”. And within that gel is a top row of “wells”. (They’ll have to think of a better word for that part though. When I think of a “well” I think of a big hole in the ground you bring water up from. These “wells” were the size of a baby fingernail cutting.) So with our precious samples loaded into the smallest of pipette tips we had to hold the pipette tip over the gel and gently squeeze out the DNA into separate wells. Four times.

You can’t let yourself get too excited or nervous. Extra shaky hands would not help in this detailed work. Once all the wells are loaded a current is run through the gel and that’s what produces that picture that makes Churchill a Bernese Mt Dog and what makes Riley and Mia Goldens.





It was an amazing day. And we did so many more things than just isolate DNA from blood. Before we even arrived Dr. Breen had taken a look at our three blood samples at the overall chromosomal level. We were shown how the chromosomes are viewed using a powerful and sophisticated microscope. We also got a VIP tour of the new hospital and a meeting with the Dean of the Vet School.










As you can see we are happy with our DNA! I can not begin to imagine what the daily running of the lab looks like. It must be a difficult and daunting task to perform these essential tests day in and day out. The professionals and students in this lab love their work. It’s evident in the passion and patience they shared with us on our day there. Beyond that, it is reflected with the information they are arming us all in the public with so that we may one day have the chance to prevent disease in our best friends and families, whether they be canine or human.








The DNA samples we isolated are loaded onto a gel in the order, Churchill, Mia, Riley. The last lane is DNA ladder used to check the size of the DNA.










Dr. Breen's Biography
http://breenlab.org/


See also this recent article
http://www.newsobserver.com/2011/06/20/1285103/dogs-help-chase-rogue-genes.html









The 8th Willem Wijnberg Cancer Fundraiser 2011

RESULTS!