ExTRacting DNA from C. elegans

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Presentation transcript:

ExTRacting DNA from C. elegans By: Brianna N. Kaylor, Joseph H. Lackey And Ryan Kelly

C. elegans 1,582 base pairs for C. elegans organic anion transporter Intestine Interested in long skinny blue intestine Grow a lot of worms Extract their DNA Looking for the transporter (organic anion transporter) www.wormbook.org 2111136

Extracting the DNA Lyse C. elegans Bind DNA to Column Wash Elute DNA from Column Detect DNA Extract DNA Spectroscopic quantification Agarose gel electrophoresis detection C. elegans In order to extract the worms to obtain the DNA, you must first wash the dish with a M9 buffer to gather as much worm as possible. Then transfer the worms to a 15mL centrifuge test tube so you can push all the worms to a pellet at the bottom. Then extract the top layer to only have worms in the 15mL tube. www.wormbook.org

Techniques Gel Electrophoresis Spectrophotometer Gel electrophoresis apparatus: Black is negative Red is positive Hooks up to power supply which pulls the DNA down the gel to the positive cation The smaller DNA will go farther down the gel Nano dropper: Can analyze a 1 μL sample of DNA or RNA Gives a graph and a concentration percentage Can analyze a 1 μL sample of DNA Use an electromagnetic field to pull the DNA from the wells to create the bands that we can see on the UV imager.

Results A260/A280 = 1.59 338 ng/nL Discovered how to use imaging and what to look for. How to make buffer Use gel Understanding how to use equipment Understand how ladders and DNA run through pores in gel

Agarose gel electrophoresis Analyzing the gel Agarose gel electrophoresis Poor separation of DNA molecules > 50 kb UV UV ethidium bromide dye is used to stain DNA

Results A single band stuck in the wells is whole, uncut genomic DNA. A smear of DNA in the lane represents large pieces of partially degraded genomic DNA, Solutions: 1)Obtain greater purity of DNA 2) Use lower agarose concentrations 3) Use enzymatic digestion to cut fragments into smaller pieces Marker DNA Marker DNA

Acknowledgements Academy of Science program Dr. Cecile A.R. Smith Department of Chemistry Dr. Tashakkori Create agarose gel Run real dna Test different concentrations of the same dna by using dyes and H2O Set up electrophoresis Dna is negatively charged Run Red