Introduction to DNA: Part 1 Genomic DNA Isolation

Lab #5 Name: ______

Introduction to DNA: Part 1 – Genomic DNA Isolation Part 2 – Mitosis and Meiosis

INTRODUCTION PART 1 - DNA is the genetic material of life. It is the blueprint that cells use to make proteins, the molecules required for almost every cellular function. A genome is the entire set of genetic instructions for a particular organism. There are regions that encode for proteins – the genes – and regions that are “non-coding” and do not have defined functions. However, the percent of non-coding regions present in the genome increases as the organism in question becomes more complex. In this lab, we will isolate genomic DNA from bacteria to have an appreciation for the wonder of DNA.

PART 2 - Cell division can be broken down into two processes, mitosis and cytokinesis. Mitosis is the process that leads to the equal distribution of DNA into the two nuclei of the two daughter cells. Cytokinesis ("cell movement") is the process that leads to the equitable distribution of cytoplasm to the two daughter cells. In short, mitosis is the division of the nucleus, and cytokinesis is the division of the cytoplasm. In this lab you will study these two processes. You will observe prepared slides of onion root tips and of whitefish blastulae, which have also been stained to highlight the nuclei. A blastula is a developmental stage in many animals. It occurs shortly after fertilization and the formation of the zygote. It is the “ball of cells” stage of development.

Meiosis consists of two nuclear divisions (meiosis I and meiosis II) and results in the production of four daughter nuclei, each of which contains only half the number of chromosomes (and half the amount of DNA) characteristic of the parental cells. During meiotic reduction of the chromosome number to half, however, chromosomes are not just divided into two sets at random. In diploid organisms, chromosomes occur in matched pairs called homologous chromosomes. These are identical in size, shape, location of their centromeres, and types of genes present. One member of each homologous pair is contributed by the “male” parent and one is contributed by the “female” parent during the process of sexual reproduction. Meiosis provides as precise a mechanism as possible for separating these homologous chromosomes so that daughter cells carry one member, or homologue, of each chromosomal pair. PURPOSE The first purpose of this laboratory exercise is to familiarize you with how genomic DNA is isolated and let you “see” the DNA. The second purpose of this lab is to investigate mitosis and meiosis by examining slides of cells undergoing each of these processes.

APPARATI AND MATERIALS PART 1 Bacterial culture Polypropylene tubes centrifuge 0.5M EDTA (pH 8.0) Lysozyme (10 mg/ml) Water bath - 37°C and 60°C 10% SDS Chloroform:isoamyl alcohol (24:1) 95% ethanol Small beakers TE (pH 8.0) 3M sodium acetate (pH 5.2) Isopropanol Eppendorf tubes

PART 2 Microscope Prepared slides of cross sections of onion root tips Prepared slides of cross sections of whitefish blastula

PROCEDURE PART 1 1. Transfer 3 mL of the dense bacterial suspension prepared for this lab into a 15 mL polyproylene culture tubes. (Note: Dispose of tips with live bacteria in bleach/water solution.) To the suspension, add 0.5 mL of 0.5M EDTA and mix the contents. Then, add 150 l of lysozyme (10 mg/mL), mix, and incubate the suspension at 37°C for 30 min with occasional gentle mixing of the tubes.

2. After incubation, complete the lysis of the cells by adding 600 l of 10% SDS and heating the mixture for 10 minutes in a 60°C water bath.

3. Allow the contents of the culture tube to cool (3 min), and then add an equal volume of 24:1 chloroform/isoamyl alcohol to the tube, stopper it securely, then shake the tube somewhat vigorously to mix the contents into an emulsion.

4. Centrifuge the emulsion at 10,000 x g for 10 min.

5. Using a pipet, transfer the aqueous phase (the upper phase) containing the nucleic acids into a small beaker; take care to avoid the precipitated proteins at the interface between the aqueous and organic phases. Slowly, stir the solution in the beaker with a clean glass rod while adding 2 volumes of 95% ethanol down the side of the beaker. The DNA will adhere to and spool around the glass rod if this is done carefully and slowly. Withdraw the rod from the beaker, and try to remove some of the alcohol from the DNA by pressing it against the side of the beaker.

6. Try to dissolve this crude DNA in 3 ml of TE (pH 8.0). When the DNA has dissolved, add 0.3 mL of 3.0 M sodium acetate. Add 1.9 ml of isopropanol and mix the contents gently but thoroughly.

7. You may be able to see the precipitated DNA at this point, but it may also be difficult to see. Centrifuge the sample at 10,000 x g for 5 minutes. Carefully remove the supernatant. Resuspend the DNA in TE buffer and transfer to a 1.5 ml eppendorf tube.

PART 2 - OBSERVING PREPARED SLIDES 1. Obtain prepared slides of cross sections of onion root tips and observe. Look for stages of mitosis. 2. Obtain prepared slides of cross sections of whitefish blastula and observe. Look for stages of mitosis. 3. Using either set of slides, sketch in the circles provided each of the phases. Note especially the spindle fibers, made of microtubules. Label the cell components visible in your drawings! 4. Using the slides of Lilium anthers and ovules observe and draw the phases of meiosis. Due to the coordination of the stages of meiosis, you will not be able to see all of the phases. Identify and draw as many as you can.

DATA AND OBSERVATIONS PART1 1. Did the bacterial culture change after the incubation in lysozyme? Did it change after the addition of SDS? What is the purpose of each of these solutions in the DNA isolation procedure?

2.What was the purpose of the chloroform:isoamyl alcohol? What does the isopropanol and the ethanol do?

Bonus: Name three techniques that can be used to analyze the genomic DNA. PART 2 Mitosis: Data from the onion root tip and whitefish blastula slides. Sketch and label each phase of mitosis that can be seen in the circles below. Note the total magnification!

Specimen: ______Phase of Mitosis: ______

Magnification: ______

Specimen: ______Phase: ______Magn: ___ __

Questions to answer: 1. Why did we look at onion root tips and whitefish blastula to study the phases of mitosis? Meiosis: Sketch and label each phase in the circles below, from Lilium anther slides. Note the total magnification!

Phase of Meiosis:

Magnification:

Questions to answer: 1. What function does meiosis serve in Lillium anthers? In what types of animal tissues would you expect to observe meiosis occurring? To Turn In: Due Thursday 7/27. Answer all questions and complete all sketches.