Restriction Digestion of Plasmid DNA using Agarose Gel Electrophoresis

Restriction Digestion of Plasmid DNA using Agarose Gel ElectrophoresisSubmitted by Group 1 Fabunan, Melody Aivi Gerardo, Mary Antonette Maguslog, Justine *Salumbre, Renz Surquia, Joseph Michael Submitted to: Ms. Gardette Valmonte Ms. Abigail Garcia October 2, 2007 INTRODUCTION Plasmids are minute genetic elements that replicate separately from the chromosome. Majority of plasmids are in the form of double-stranded DNA (dsDNA) 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 have inherent antibiotic resistance genes and are 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 double-stranded 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 homodimer and Mg2+ alone is involved as a cofactor. The cleavage site is defined and within the recognition site, leading to a 3’overhang, 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 pGEX 4T1 with restriction enzymes. MATERIALS AND METHODS The materials utilized in this experiment are plasmid pGEX 4T1; restriction enzymes which are BamHI, EcoRI, HindIII and Pst I; restriction enzyme buffers, micropipettors; 0.5-mL microcentrifuge tubes; pipette tips and crushed ice and Styrofoam cup. For the Agarose Gel Electrophoresis, the materials are restriction digests; 10X Gel loading dye; 1% Agarose in 1X Trisacetate-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. For the methodolody part of the experiment, eight 0.5-ml microtubes were prepared for each tube: the first microtube was assigned for the Bam HI, for the second tube, EcoRI and Sal I, third, Bam HI and EcoRI, fourth, EcoRI, fifth, BamHI and Sty I, sixth, Sal I, seventh, Sty I and Pst I, and lastly, eighth, Bam HI, Hind III, and EcoR I. And to each tube, 5 µL of plasmid pGEX-4T-1, 1 µL of appropriate 10X restriction enzyme and 1 µL of appropriate 10X restriction enzyme were added in sequence. They were then subjected in a microcentrifuge so that the samples, once settled in the bottom, may be collected. The tubes were then incubated at 37OC. Consequentially; 4 µl of gel loading dyes was added to each microtubes. 10 µL from each microtubes were loaded and subjected to the analysis of restriction fragments thru the Agarose gel electrophoresis. 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 µL 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 µL of each sample were loaded into separate wells in the gel chamber. 10 µL 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. RESULTS AND 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 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. The assigned restriction enzyme for Group 1 is BamH 1. According to the Promega website, Bam HI has a minimum of two units necessary to cut 1µg 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 Group 1 Figure 1. Result of Agarose Gel Electrophoresis Figure 2. Plasmid Map of pGEX 4T1 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 http://www.wikipedia.org