Briana Halbert Restriction Enzyme Digestion of DNA and Gel Electrophoresis November 8, 2012 Purpose The purpose of this

experiment is to understand what a DNA restriction enzyme is and how it works. Learn to separate DNA on an agarose gel using electrophoresis. Understand how to use a restriction map to identify a sample DNA. Reagents Reagent CH3CO2K (Potassium acetate) Mg(CH3COO)2 (Magnesium acetate) C10H16N2O8 (EDTA) C12H20BrN3 (Ethidum Bromide) Molecular Weight 98.15 g/mol 142.394 g/mol 292.24 g/mol 394.33 g/mol Melting Point 292C 80C -144.9C 239C Boiling Point 758.8C N/A N/A 261C Density 1.57 g/cm 1.45 g/cm 860.00 kg/m3 N/A Hazard Irritant Irritant Irritant Irritant

Introduction Plasmids are minute genetic elements that replicate separately from the chromosome. Majority of plasmids are in the form of double-stranded DNA and may either be circular or linear. Plasmid DNA is usually employed in recombinant DNA technology to clone a specific segment of DNA resulting most advantageously in that large quantities of these segments can be prepared. Moreover, plasmid DNA has inherent antibiotic resistance genes and is used to introduce genes into cells by transformation. Restriction enzymes, known also as restriction endonucleases, recognize short DNA sequences which are often palindromic. These enzymes cleave doublestranded DNA at specific sites within or adjacent to their recognition sequences. Restriction enzymes have specific requirements needed for optimal activity of the said enzymes. As such, certain conditions such as temperature, pH, enzyme cofactors, salt composition and ionic strength affect enzyme activity and stability. There exist three types of restriction enzymes: Type I have bipartite and interrupted recognition sequences; and its restriction activity is usually a pentameric complex. The cofactors and activators involved in this type Mg2+, AdoMet and hydrolyzed ATP. The cleavage site is distant and variable from recognition site. Common examples are EcoK I, EcoA I, EcoB I, CfrA I, StyLT III and StySP I. Type II have recognition sequences which are either palindromic or an interrupted palindrome (giving way for ambiguity). Its subunit structure is a 1

Briana Halbert Restriction Enzyme Digestion of DNA and Gel Electrophoresis November 8, 2012 2+ homodimer and Mg alone is involved as a cofactor. The cleavage site is defined and within the recognition site, leading to a 3overhang, 5 overhang or blunt end. Common examples are EcoR I, BamH I, Hind III, Kpn I, Not I, Pst I, Sma I and Xho I. Lastly, Type III enzymes have non-palindromic recognition sequences and the cofactors and activators involved are Mg2+, AdoMet, ATP (not hydrolyzed). The cleavage site cuts approximately 25 bases away from the recongnition sequence and may not cut to completion. Common Type III enzymes are EcoP15 I, EcoP I, Hinf III, and StyLT I. Agarose Gel Electrophoresis is a useful tool for the rapid separation of DNA fragments. Electrophoresis is usually involved in the monitoring of enzyme reaction, resolution of DNA fragments for transfer to membranes and hybridization, or preparation of fragments for labeling etc. The objectives of this experiment are the following: to electrophorese the digests on an agarose gel and to determine the sizes of the DNA fragments generated from digestion of the plasmid with restriction enzymes. Procedure For the Agarose gel electrophoresis, the first phase consisted of preparing the gel. A submarine mini-gel was set up and 1% agarose was melted in 1X TAE buffer in the microwave oven. The melted agarose was cooled and poured into the gel-casting tray. The comb was put in and the gel was allowed to set for 30 minutes. The second phase concerns the preparation of the samples. For this phase, the digested DNA were taken out of the refrigerator. 2 L of gel loading dye was added into each tube. The contents were mixed and subjected under the microcentrifuge. Once the gel has set, the gel and tray was placed into the electrophoresis chamber thus initiating the sample-loading and running of the gel phase. A sufficient amount of 1X TAE buffer was poured such that the gel was completely submerged with the wells filled up. 10 L of each sample were loaded into separate wells in the gel chamber. 10 L of the DNA size markers were also loaded in the first lane. The lid was placed on the chamber and the electrical leads were connected. The power was turned on and the gel run at about 100 volts. The last phase of the electrophoresis part is the staining and destaining of the gel. Once electrophoresis was completed, the gel and tray were removed from the chamber. The gel was slid into a staining tray containg 200 mL of distilled water and 2 drops of ethidium bromide. The gel was then stained for 20 minutes and the ethidium bromide was removed and contained in an amber bottle. 200 mL of distilled water was then added and the gel destained for 10 minutes. The water used was discarded into a waste bottle. For the purposes of visualization, the gel was subjected under the UV transilluminator. The materials utilized in this experiment are plasmid pGEX 4T1; restriction enzymes which are BamH1 and Nde1; restriction enzyme buffers, micropipettors; 0.5 mL 2

Briana Halbert Restriction Enzyme Digestion of DNA and Gel Electrophoresis November 8, 2012 microcentrifuge tubes; pipette tips and crushed ice. For the Agarose Gel Electrophoresis, the materials are restriction digests; 10X Gel loading dye; 1% Agarose in 1X Tris-acetate-EDTA buffer; 1X Tris-acetate-EDTA buffer prepared from 40X stock solution; Ethidium bromide and DNA size markers. The special equipment used are the microcentrifuge; agarose gel electrophoresis set-up with power supply, transilluminator and a digital camera. Results The assigned restriction enzyme for is BamH 1 and Nde1. According to the Promega website, Bam HI has a minimum of two units necessary to cut 1g of supercoiled DNA containing a single restriction site. If the Bam H1 cuts, there should be only one fragments and the size remains to be 4969bp.
DNA Ladder

Figure 1. Result of Agarose Gel Electrophoresis

Briana Halbert Restriction Enzyme Digestion of DNA and Gel Electrophoresis November 8, 2012

Figure 2. Plasmid Map of BamH1 and Nde1

Discussion Electrophoresis, in this case Agarose gel electrophoresis, was used to resolve the mixtures of plasmid DNA fragments, specifically, restriction enzyme-digested genomic DNA. This mixture contains millions of different fragments once a restriction enzyme has been applied. Fragments have been visually identified by use of the UV transilluminator. Furthermore, gel electrophoresis has been used to purify DNA fragments from other fragments of various sizes. These fragments are synthesized by restriction enzyme digestion of the cloned DNA and resolved by agarose gel electrophoresis. In the field of experimental molecular biology, a supercoiled plasmid DNA is cut at a single site within multiple cloning sequences. The ability of a restriction enzyme, according to the Promega Restriction Enzyme Resource, with the

Briana Halbert Restriction Enzyme Digestion of DNA and Gel Electrophoresis November 8, 2012 consideration of finding a single site by linear diffusion in the supoercoiled plasmid is different than for any of the sites in a linear substrate. Moreover, some enzymes have exhibited great differences in their ability to cut plasmid DNA. These are usually dependent on buffer conditions. Questions 1. What is the purpose of adding a buffer to your enzyme reaction? To resist changes in pH that may occur during this reaction. And also to digest enzymes simultaneously. If a suitable buffer in which all your enzymes function properly you will have to make a buffer exchange. 2. Why would your DNA sample run from the negative electrode to the positive electrode? DNA is a negatively charged molecule due to the phosphate groups that constitute the backbone of DNA. Therefore, by placing the DNA at the negative end of the gel matrix, when the current is turned on the DNA will migrate down the gel towards the positive side because the opposite charges attract. 3. What is the purpose of adding a loading dye to your DNA sample before it is subjected to gel electrophoresis? If you do not add this dye to the gel before you pour it into the red striped tray, you will NOT be able to see the DNA. 4. Write a 6-base pair palindromic sequence. Please include both strands. GATATC CTATAG 5. Explain the difference between a blunt cut and a staggered cut. Restriction exonucleases can be seen as restriction enzymes that cleave by blunt cuts because they cut sequences at the ends. Staggered cuts have a zig zag conformation and result in sticky ends.

6. Why did you incubate your enzyme/DNA mixture at 370C for one hour. So that the Enzyme did not degrade and also because most human enzymes react the "fastest" at human body temperature, which is what they were "designed" for, which is 37 degrees. 7. Explain how ethidium bromide stains DNA. Ethidium bromide interculates with DNA, i.e. it slips in between the base pairs and stays there. It also fluoresces under UV light so you can see the location of the DNA (e.g. on an agarose gel). Ethidium bromide is also mutagenic, it causes damage to DNA, exposure to it (skin contact or inhalation) should be avoided as it will damage the DNA in the cells it comes into contact with and hence have a negative effect on the cell.

Briana Halbert Restriction Enzyme Digestion of DNA and Gel Electrophoresis November 8, 2012 References Davis, L.G., W.M. Kuehl & J.F. Battey. Basic methods in molecular biology (2nd ed.). Appleton & Lange : Norwalk, Connecticut, USA. 1994. Department of Biological Sciences. Laboratory Protocols in Cell and Molecular Biology. UST: Manila. 2007. Karp, G. Cell and molecular biology: Concepts and experiments. John Wiley & Sons : Asia. 2008. http://www.promega.com