Restriction Enzyme Digestion of PCR Product and Agarose Gel Electrophoresis
Today you will digest your PCR products with one restriction enzyme (EcoRV) and then visualize the DNA fragments of both digested and undigested pcr products by performing electrophoresis on a 1.5% agarose gel.
1. Label the tops of four (or however many putative transgenic plants you have to test) microfuge tubes with your initials or team color and the plant number. (You will digest each of the putative transgenic plants for which you have pcr product from our gusA amplification; however, you will NOT digest the non-transgenic control plants.)
2. Set up the following digests, adding the reagents to the microfuge tube in the order they are listed.
10 microliters of PCR product (or half of it)
7 microliters of water
2 microliters of salt buffer (100 mM NaCl, 50 mM Tris-HCl, 10 mM MgCl2, 1 mM DTT, 100 ug/ml BSA, pH 7.9)
1 microliter of restriction enzyme EcoRV
3. Close the tubes and flick to mix the contents, then give the tubes a quick pulse in the microfuge. Put the tubes in the 37°C heatblock for 30 minutes.
4. At the end of the incubation time, give the tubes a quick pulse in the microfuge to bring down the condensation that is on the top of the lid. You can then open the caps and add 3 microliters of loading dye to each digest (the undigested pcr products already have sufficient loading dye). Change tips between additions. Close the caps and flick to mix the contents, then give the tubes a quick pulse in the microfuge if there are any droplets of liquid on the wall of the tubes. (If you need to pulse the undigested pcr products to get all the contents to the bottom of the tubes, you will have to put the pcr tubes in an adapter before spinning or just shake them to get the contents to the bottom. Be careful with the pcr tubes. They are thin walled and can break.)
5. Keep your digests and your undigested pcr products in your ice bucket until your instructor tells your class to start loading. Sign up for lanes on the gel templates provided by your instructor. Each group will load a 1.5% agarose gel containing SybrSafe™ stain with 1), all of the undigested pcr product from their non-transgenic control (in a separate area on one of the gels as indicated on the template--no need to add more loading dye)) and for each putative transgenic, 2), the rest of your undigested PCR product (no need to add more loading dye) next to 3) 23 microliters of digested pcr product for that plant.
When you load the gel, be sure to use only the lanes on the template that are assigned to your group . If you make a mistake, be sure to revise the template. Your instructor will add to one well on the gel the molecular weight standards. Be sure and pick up a key for the standards ladder or check with your instructor to see if she is going to label the standards with the base pair numbers before she posts the gel photo to the data file.
Once all the samples are loaded, the current (~100V) will be applied to the gel for one hour. The gel will then be photographed under UV light, the lanes labeled and your instructor will post the gel photos to the data file on the lab conference.
Once you have decided whether or not there is sufficient evidence for you to conclude that the gusA gene is present or absent in that tobacco clone or control, please fill out the last column on the course spread sheet with 1 for positive or 0 for negative to score each of your group's plants (including the non-transgenic control).
How do you know what size DNA fragments to expect from your PCR amplification of gusA and the restriction enzyme digestion of this gene with EcoRV?
There are several FREE online services to help us out. My favorite is New England Biolabs.
- Click on the above link to reach their site.
- At the bottom of the page click on NEB cutter
- On this page you can enter the specific DNA sequence of our PCR product. To do this - open the WORD file of Media:GUS A SEQUENCE IN pBI121 1812 bp.doc. Then highlight the region including and between the two underlined primers. Copy it and paste it into the box on the NEB website.
- Make sure the circle next to NEB enzymes is filled in (should be default). Click SUBMIT.
- The next page will bring you to a linear piece of DNA cut with lots of different enzymes. How big is your PCR product? We only care about one - EcoRV - so click on Custom Digest on the left hand side of the screen.
- Scroll down the list of enzymes until you find EcoRV. Click the check box. And press the green Digest button at the bottom of the screen.
- The next screen should have your piece of DNA cut with EcoRV. How many unique places does EcoRV cut?
- Hover over each site with your mouse and it will tell you the exact base pair the enzyme cuts at. Take note in your lab notebook.
- Now click on the View Gel button on the left hand side of the screen.
- Take note of the expected band sizes at the top of the screen - how many are there? Look at the gel - how many bands do you see?
This is a nice comparison for the gel you are running today.
To be discussed while our gel runs
Apse, G.P., Aharon, G.S., Snedden, W.A., Blumwald, E. "Salt Tolerance Conferred by Overexpression of a Vacuolar Na+/H+ Antiport in Arabidopsis" Science (1999) 285: 1256-1258. Available at: http://www.sciencemag.org/cgi/content/full/285/5431/1256
or in pdf form at http://0-www.sciencemag.org.luna.wellesley.edu:80/cgi/reprint/285/5431/1256.pdf
There is also a Perspectives article associated with this paper: Perspective
Uddin MI et al., "Overexpression of a New Rice Vacuolar Antiporter Regulating Protein OsARP Improves Salt Tolerance in Tobacco". Plant and Cell Physiology (2008), Volume 49, Number 6 Pp. 880-890. Available at: http://pcp.oxfordjournals.org/cgi/content/full/49/6/880
A Feb. 5, 2002 Boston Globe article by ME Malone,
SCIENTISTS FOCUS ON THE TOBACCO PLANT AS A POSSIBLE CANCER-FIGHTER GENETICALLY ALTERED CROPS MAY SOMEDAY PRODUCE DRUGS TO COMBAT MANY DISEASES. Available from Lexus/Nexus Academic http://0-www.lexisnexis.com.luna.wellesley.edu/us/lnacademic/search/flap.do?flapID=news&random=0.7807457835138732