Ag Experiment #1 Spooling DNA
(This experiment was copied from the April issue of "Making A Difference" publication. The experiment was written by Lisa Konkel, Big Foot Union High School Agriscience Instructor.)
(Note: three options are provided, beef liver, calf thymus or E.coli bacteria. You can complete one, or all three and compare the results.)
- Extracting DNA from an animal or bacteria cell
- Spooling DNA
- Separate and collect the DNA from E. coli bacteria, beef liver or calf thymus.
- Describe the appearance of DNA extracted from a cell.
- Relate the location of DNA in a cell to procedures for extracting it.
- Verify the DNA is an acid.
Spooling Beef Liver DNA Lab
(Time needed to complete this lab. 45-50 minutes.)
- 2 cm piece of beef liver (can be acquired from a grocery store)
- mortar and pestle
- fine sand
- SDC/NaCl solution
- test tube
- ice water
- inoculating loop
(Note: SDS solution can be substituted with Woolite detergent.)
- Put liver piece in mortar with a couple pinches of sand.
- Add 10 ml of SDS/NaCl to the mixture and grind with pestle until mixture forms a thick fluid.
- Place several layers of cheesecloth in a funnel and pour mortar contents through it to gain 2ml of extract in a test tube. (note: you might have to squeeze the cheesecloth)
- Put test tube in an ice water bath.
- Pour 4 ml of ice-cold ethanol into the tube. As with the E coli lab, pour very slowly and at a 45 degree angle.
- Rotate inoculating loop slowly at interface area and see the DNA results.
Spooling Calf Thymus Lab
(Time needed to complete this lab: 30 minutes if you blend ingredients first, 45-50 minutes if you do all procedures in class.)
- calf testes
- 95% ethanol
- Adolph's Meat Tenderizer (from grocery store)
- wooden stir rod (skewer sticks purchased at the grocery store work well)
- Place 100 ml of water with a 2cm testes piece in a blender and blend until smooth.
- Pour mixture into a beaker, add 5 ml of Woolite and stir.
- Pipette 5 ml of solution into each student's test tube.
- Add 3 grams of Adolph's Meat Tenderizer and stir. (3 grams is just enough to cover the tip of a spatula.)
- Place tubes in 60 degree water bath for 15-20 minutes.
- Pour 5 ml of cold ethanol slowly down the tube at a 45 degree angle.
- Gently insert the wooden stir rod and rotate to spool DNA.
E. coli Lab
Time needed to complete this lab: approximately 45-60 minutes (based on cooling time required)
- 6 tubes freeze dried E.coli
- E.coli suspension medium
- DNA suspension medium
- Sodium Dodecyl Sulfate
- Alkaline pH indicator
- Spooling rods (wooden skewer sticks from the grocery store work well)
- Graduated plastic pipettes
Note: a kit can be ordered from Carolina Biological 17-1090, includes addition background information and student questions.
- Measure 5 ml of E.coli suspension medium in a small tube or cup and put it in the tube of freeze dried E.coli bacteria. Cap the tube and shake it slowly so the bacteria dissolves in the solution.
- Take 1.0 ml of sodium dodecyl sulfate (SDS) and add to your bacteria tube. Swirl the tube slowly. This process breaks apart the cell walls (lysing) and allows the DNA to be released.
- Put tube in a 65 degree C hot water bath for 30 minutes, then cool to room temperature.
- During the 30-minute wait, add .5 ml alkaline pH indicator to 10 ml of 95% ethanol, then refrigerate until needed.
- Place your stirring rod in the tube. Add ethanol mixture to cooled tube.
- IMPORTANT!! Add this mixture very slowly and have the tube tilted at a 45 degree angle. This is to prevent the ethanol and suspension medium form mixing. Note any color change at the liquid interface.
- Continuing at a 45 degree angle, rotate the rod in the same place over and over. Visible string-like strands should twist onto the rod at the interface level.
- Once you have a visible sample on the rod, remove it and place in 95% ethanol for two minutes, then air dry for five minutes.
- Place rod in 10 ml of DNA suspension medium, wait 20 minutes and look for color change.
It is very helpful for students to be able to see DNA strands because it makes it "real". They begin to really believe that it exists. For my classes, I complete all three labs and have the students compare the structure of their results--the viscosity, color, texture, etc. This proves that different living organisms all start with DNA. If some teachers find that they cannot work all three into their budget or don't have the materials on hand, the basic principle can easily be accomplished by completing only one of the labs.