1 Restriction Digestion of Plasmid DNA and Gel Electrophoresis 2007-08 INS Winter Quarter – Lab 3 Goals: You will learn to a) work with DNA b) use DNA modifying enzymes and c) separate DNA fragments by size using agarose gel electrophoresis. To enhance this experience, it is advised that you complete the prelab worksheet provided at the end of this manual. Restriction enzymes are produced by bacteria to prevent foreign DNA and virus sequences from surviving in the cell. These are endonucleases, meaning they cut both strands of DNA at some location other than the end. (The counterpart to this would be an exonuclease, which would digest a DNA molecule starting from an already cut end.) The value of many restriction enzymes is that they cut at specific base sequences and nowhere else. These recognition sites are usually palindromes and can be from 4 to 15 bases in length. The two enzymes we will use today are SacII (Streptomyces achromogenes, second (II) enzyme isolated) and XhoI (Xanthomonas holcicola, first (I) enzyme isolated). The XhoI enzyme cuts (indicated by the arrow) at the sequence C▼TCGAG, while SacII cuts at the sequence CC GC▼GG. Today you will be cutting the 5601 base pair (bp) plasmid, pCDNA3.1Hygro+, with these enzymes. The plasmid contains one recognition site for each enzyme. The XhoI enzyme cuts at nucleotide 986 whereas the SacII cuts at nucleotide 2900 on the plasmid. To determine the number and lengths of the product DNA fragments, we will use agarose gel electrophoresis. At normal pH values, DNA molecules carry a large number of negative charges -- they are polyanions. If the DNA molecules are placed in an electric field, opposite charges attract so the DNA will move towards the positive electrode and away from the negative electrode. This technique of separating molecules by their movement in an electric field is called electrophoresis. Since the number of negative charges on a DNA molecule increases in proportion to its mass, the acceleration on all molecules is roughly the same. To help prevent the molecules from moving, this separation is done in a porous gel. Agarose is a highly purified carbohydrate obtained from kelp. A 1-2% mixture of agarose creates an easy to handle gel. As the DNA molecules are moved through this gel, the smaller molecules pass more quickly while the larger molecules become hung up on the gel and move more slowly. By combining the driving force of the electric field with the varying drag of the agarose gel, a mixture of DNA fragments will separate out by size. As a rough approximation, a plot of distance moved (x-axis) versus log [molecular weight] (y-axis) produces a close to linear line. To help identify the sizes of the fragments, standards (DNA ladder) will be included with our experimental samples on the gel. By using the known fragments provided by these standards, you can calibrate your agarose gel and prepare a reasonable size estimate of the DNA fragments produced in your restriction enzyme digestions. NOTE: Many restriction enzymes do not last very long in warm conditions. You should develop the habit of keeping them as cold as possible and minimize their exposure to high temperatures. Use a an ice bucket and keep enzymes on ice until needed. Do not warm the enzymes up in your hands! SAFETY: Ethidium bromide is a mutagen. Always wear gloves when handling any material containing ethidium bromide and dispose of it in the proper location. Any gloves worn when handling ethidium solutions should also go in the designated waste. Any person in the same room as the UV light should wear UV-filtering eye protection any time the light source is on. Not all safety glasses are approved for UV protection. Check the label on yours or use one of the pairs of goggles provided. Know what you are doing when working with the power sources. Large pieces Small pieces DNA runs toward positive electrode2 Procedure: I. Digest DNA samples: (We will provide exact volumes on the day of the lab.) 1. Label your reaction tubes. Each pair will do 3 enzyme reactions of plasmid DNA. Your plasmid will be separately digested with XhoI and SacII, and also digested with both enzymes. Place the labeled tubes on ice. 2. Using a clean tip for each sample, transfer ___ µl DNA of each sample into the appropriate tube. 3. Obtain the small tubes of the enzyme mixes. These contain restriction enzyme and a buffer mix that adjusts the pH and salt concentration to the correct level for these enzymes. THESE SHOULD ALWAYS BE KEPT ON ICE. 4. Using a fresh pipette tip for each sample, transfer ___µl of the enzyme solution to each of the DNA samples. If you feel there is any chance of a tip being contaminated or touching the wrong solution, replace the tip. NOTEBOOK CONTENT: A table describing the contents of each tube is handy here. It also helps to check off the additions to each tube as they are made. 5. Close the tops and mix the sample. Flick the bottom with your finger several times and then briefly run the sample in a microcentrifuge. 6. Incubate the samples at 37° for 1 hour. During this incubation period, you should familiarize yourself with the electrophoresis apparatus, pour your agarose gel, and practice loading samples. When the time period is up, stop the reaction by adding loading buffer (step 4 in electrophoresis directions) and putting your sample on ice. II. Practice loading a gel We will have an agarose gel for you to practice your loading technique before you use your real samples. Please use the 1X dye sample we provide to familiarize yourself with the process. III. Electrophoresis of samples: 1. We will demonstrate how to pour an agarose gel for you to use in this lab. (The gel consists of 1% agarose in 1X TAE buffer (40 mM Tris-acetate (a buffer) and 1mM EDTA (a metal binder), pH 8.2-8.4). This mixture was heated until all of the agarose was dissolved and then kept in the oven to stay liquid. Use the 8 well comb when pouring your gel. 2. After the gel solidifies, place it in the gel box. The wells in which the samples will be loaded should be at the (-) end of the box, where the black lead is connected. 3. Pour electrophoresis buffer TAE into the box until it just covers the agarose. 4. Using a clean tip for each of your samples, add loading dye to each restriction digest tube to a final concentration of 1X. BE SURE NOT TO CONFUSE THE SIZE STANDARDS WITH THE LOADING DYE. Mix the loading dye and your sample. Also do