MadSci Network: Molecular Biology |
This sounds like it’s going to be a very visual experiment. Using DNA from onion, peas, lamb and chicken liver should yield some very interesting banding patterns when all of your samples are digested with the same restriction enzyme. Restriction enzymes, also known as endonucleases, are enzymes that recognize very specific “target sequences” in DNA and then lyse, or cut, the DNA at that site. For example, the endonuclease EcoRI, derived from E. coli, recognizes the sequence GAATTC and will cleave the DNA after the 5’guanine on each strand, in the following manner: 5’-- G/AATTC --3’ 3’-- C TTAA/G --5’ Every time the enzyme encounters this sequence, the DNA is cut apart. The number and size of the DNA fragments will depend upon how many times this sequence occurs within the genome and how many basepairs are in between each sequence. Make sense? Since your experiment focuses on the differences in the DNA of four different species of both plants and animals, your choice of enzyme is not critical, so I would suggest using EcoRI, since it is a very common enzyme and is not too expensive, but for a list of other enzymes and their target sequences, you can click here. The buffer you will use will come with the enzyme when you order it, but be aware the buffer comes as a 10X stock solution, so you will need to dilute it to 1X before you use it. Your question about how much DNA to load on a gel is a critical one and shows you are thinking! Oftentimes students think that more is better, but you should remember the Goldilocks standard: Too much DNA and your gel will look like one big smear. Too little DNA and your bands may be too faint to see. You need to load your gel JUST RIGHT. In general, if you’re using a miniprep kit to extract your DNA, you should use about 4µL of extracted DNA, 1µL of EcoRI and 15µL of 1X buffer in your digestion reaction. After the incubation, just add 2µL of loading dye and load 20µL onto your gel. Methodbook.net has a great detailed explanation of this and a standard protocol. You should also bookmark Protocol-online.org. This is a great resource to search for any type of protocol you might need now and in the future. Your final question is one that YOU need to think about: “. . . what can be determined from the differences seen in the bands?” Agarose gel electrophoresis separates the DNA fragments according to size, with the smallest fragments traveling furthest through the gel. From what you know about how DNA differs (or is similar) between species, what would you expect to see? Do you think the plants will have similar bands? Do you think there will be more similarities between lamb and chicken than there will be between lamb and peas? What about lamb and onion? Or do you think that all four phenotypes (physical characteristics) are so different that you expect no similarities between their genotypes (genetic makeup)? I can think of several good hypotheses you can formulate about what similarities and differences you expect to see. After you run your gel, compare each pattern to see if they support your hypotheses. Have fun! Sue Baker, Mad Scientist P.S. If you get really good results, try this: Prepare another digestion of all of the samples again, but this time have your teacher prepare an extra tube of one of the samples at random. This should be labelled “unknown.” (Only your teacher knows its true identity.) Then run all five samples side-by-side on a gel and see if you can identify the mystery DNA by comparing its banding pattern to all of the knowns.
Try the links in the MadSci Library for more information on Molecular Biology.