cell biology lab report on LAB 1A: DNA Isolation from Wheat Germ LAB 1B: Spectrophotometric Quantification of DNA and Protein

In order to isolate biomolecules, three basic steps must be achieved. First, the tissue or cells must be broken apart (homogenized) in order to release the molecule of interest. The homogenate is then centrifuged in order to sediment the insoluble material into a pellet and produce the supernatant, which contains any soluble molecules. Depending upon the molecule of interest, the pellet or supernatant is then treated further in order to isolate and enrich the biomolecule in the final sample. Various physical (electrophoresis, filtration) and chemical (pH, ionic strength, hydrophobicity) methods have been developed to allow us to perform the isolation and enrichment of different biomolecules.

In this lab, we will perform an isolation and enrichment protocol to obtain DNA from wheat germ . A figure showing the components of a grain of wheat are shown in Figure 1 . The wheat germ is particularly high in DNA content, relative to proteins and lipids, and is therefore preferred for this experiment.

Figure 1: A kernel of wheat. The wheat germ or “embryo” is used for the purpose of DNA extraction. Adapted from: https://live2eatlovelaugh.com/recipe/soft-wholemeal-bread-recipe/ Bran: is included in whole wheat flour. It is rich in B-complex vitamins and dietary fibre Endosperm: is primary component of white flour. It is largely comprised of starch.

Germ: not included in white flour preparations due to the high fat content. This includes the “embryo”, and is also rich in Vitamin E. PROCEDURES Note: the times indicated for this experiment are required to ensure proper mixing at each stage and eventual isolation of DNA. Please use the timers provided on your bench top.

5 grams of wheat germ have been weighed and then placed in the blender container on your bench.

1. Pour 30 mls of warm DNA buffer (0.14 M NaCl and 0.01 M sodium citrate) into the blender container, and fit the container to the blender base. Place the cover on the container, and blend at high speed for about 1 minute and then turn off the blender. Wait 15 seconds , then blend at high speed for another minute . Nuclei and DNA have been released by the blending action. The DNA buffer contains sodium citrate to help prevent any DNAase activity. Citrate ions chelate Mg 2+ , an ion that is essential for DNAase activity.

2. Split the entire homogenate mixture into two 50-ml centrifuge tubes and balance them . Place the tubes opposite each other into the rotor, and centrifuge the mixture at 4 o C for 10 minutes at 4,000 x g. Remove the centrifuge tubes from the centrifuge rotor, being careful not to disturb the pellets , which contains the all important nuclei and DNA needed for the next steps in the procedure. Discard the supernatants by slowly pouring into a beaker. Be careful: sometimes the pellet gets dislodged at this stage, and starts to slide out of the tube.

3. Pour 5 mls of warm DNA buffer and 4 drops (which is about 0.4 ml) of TRITON-X into each centrifuge tube . You will need to dislodge the pellet and break it up into smaller pieces with the plastic rod. Then s hake the bottle vigorously for 5 minutes. This shaking will help resuspend the pellet, and the Triton-X, which is a hydrophobic detergent, will help to dissociate the DNP complex as well as dissolve any residual membrane debris present. Continue shaking if the mixture does not look relatively homogeneous. Combine your sample into a single tube.

4. Prepare a balance tube with water and centrifuge this mixture at 4 o C for 15 minutes at 4,000 x g . Discard the supernatant .

5. Measure 30 ml of 2.6M NaCl into a graduated cylinder, and pour approximately half of the NaCl solution into the centrifuge tube. You will need to dislodge the pellet and break it up into smaller pieces with the plastic rod. Add the remaining NaCl solution. DNA is soluble in this high salt solution, so if the pellet is vigorously agitated by shaking for 5 minutes, the DNA will gradually dissolve, whereas the proteins will precipitate. You should notice that the suspension becomes quite syrupy (viscous) as you shake it. This viscosity is due to the long fibres of DNA.

6. When you are satisfied the pellet has been well suspended, again prepare a balance tube with water, and centrifuge the mixture at 4 o C for 15 minutes at 20,000 x g.

PLEASE NOTE: BE SURE THE TUBES ARE BALANCED. AT THIS SPEED UNBALANCED TUBES CAN SERIOUSLY DAMAGE THE CENTRIFUGE!

2 7. Carefully pour the supernatant into a clean 100-ml beaker . As before, make sure that the pellet does not become dislodged. SLOWLY add 30 mls of 70% ethanol to the beaker by carefully pouring it down the inside wall of the beaker containing your supernatant, so that you obtain a two-phase solution : the ethanol on top, and the DNA-containing salt solution below. At the interface of the ethanol-salt solution, you will see strands of DNA precipitating in the ethanol solution. Allow the DNA to precipitate for 5 minutes .

8. Label a plastic Petri dish (DNA does not stick to plastic, so plastic dishes are much better than glass dishes) with your group number and lab time. Take a plastic fork and rapidly swirl it through the solution to collect the DNA. When sufficient material collects on the fork, remove it by scraping it with a needle probe or cutting it with a razor blade into the plastic Petri dish . Collect as much of the DNA out of the solution as possible. What does the DNA look like at this point?

9. Use a Pasteur pipette to remove as much residual liquid as possible from the Petri dish containing your collected DNA. Then wash your DNA by adding 10 mls of 70% ethanol to the Petri dish and swirling it a few times. Concentrate your DNA into a single quadrant of the Petri plate and use a Pasteur pipette to remove as much residual liquid as possible .

Place the Petri dishes of "wet" DNA on the labelled shelf at the back of the lab. Leave the lid off the petri dish and use it to prop up the dish at an angle so that any excess liquid can continue to drain and the DNA will dry. This will be the wheat germ DNA preparation that you will use in Lab 1B for both DNA and protein concentration determination.

Please view the Centrifugation video prior to the Lab 1A Tutorial . The Centrifuge Use Guidelines are available in Appendix A .

There is no assignment following this week’s lab. However, the procedures employed must be included in your formal Lab 1 Report , which will be due on October 8 th by 11:59 PM. This report is worth 7.5% of your final grade and must be submitted as a pdf through Nexus.

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