Restriction Endonuclease Digestion of Plasmid Dna

Introduction With the execution of this essay, we began to go deeper into the Cell and Molecular biological science course. The main focus of the try out would be how the restraint Endonucl moderations stupefy the strands of deoxyribonucleic tart. For this experiment, pBR322 was the specimen to theatrical role. obstruction Endonucleases work by cleaving the sugar phosphate backb mavin of specific DNA sites. limitation enzymes that nourish been isolated from bacteria have a defensive role. This idea is illustrated when an attack foreign cell DNA is difficult to alter the bacteria parapet enzymes cleave the DNA rendering it inert.The second variance of the experiment deals with gel cataphoresis. The samples are nasty into wells on an 1% agarose slab and subjected to galvanizing currents both positive and negative. Our current signal here is DNA, therefore since nucleic acid as a negative charge, the bands will immigrate toward the positive cathode. This process of migration is called sieving and smaller strands hunt down faster than longer strands due to their ease in going through the gel. The objectives of the experiment embarrassLearning the principles behind Restriction Enzymes and colloidal gel Electrophoresis Applying the concepts in the experiment to produce bands at the end of the Gel Electrophoresis stage Interpreting what these bands conceive with accordance to how the plasmid was cleaved Methods and Materials For the experiment we use several labour endonucleases (BamHI, EcoRI, HindIII, PstI, ScaI, SaII), ppBR322 plasmid DNA, TAE/TE Buffer, DNA Ladder (50 Bp), Restriction Buffers, 1g of Agarose, 700ml of Distilled H2O. Equipment utilize for the experiment included Agarose Gel Electrophoresis System, Uv-vis illuminator and Camera or a Gel doc-it documentation system.The first procedure began by adding 8. 5 L sterile distilled H2O, 1. 0L of the appropriate 10x buffer, 1. 0L disentangleination of the restriction endonucleases an d 1. 0L of pBR322 plasmid DNA (the DNA would be added last) in 5 separate 1. 5ml microcentrifuge tubes, one tube is not to have an RE in it. The mixture was so incubated for 1 hour at 37 C. No dry block heater was ready(prenominal) so body heat was used. after(prenominal) incubation, 2L of gel dispatch blot (Bromphenol Blue) was added to each mixture and nonsensical in 1% agarose gel. The 50bp DNA lead was placed in lane 1.It was because subjected to electrophoresis at 100V 250mA 50W. Agarose gel was on the watch by dissolving 1g of agarose gel demolish in 100mL distilled H2O in a microwave over. It was then(prenominal)(prenominal) cooled at 60C then poured in a gel casting tray. A comb was then put and the gel was leftover to solidify. Afterwards, the gel casting tray was placed into the bomber gel electrophoresis system. The TAE buffer was then placed. The samples were then loaded from left to unspoilt starting with the DNA ladder on lane with and the sample without any restriction enzyme on the extreme right.It was then cover and the anodes were attached on the side of the walls. They were connected to the power run set at 100V 250 mA 50W and then run. When the tracking dye reached near the end point, the power supply was turned off. The gel was then remove and ecstasyred into a developing try containing a 10L ethidium cliche pero 100ml buffer. It was then shook for 15 minutes. The get was then transferred to the documentation system and Rf determine were measured. Pictures were taken and the gel was immersed in hypocholorite (chlorox) response before discarding. Results and DiscussionThe group did not include a mix without restriction enzymes because doing so will lead to undigested or in all told digested DNA. The DNA methyltransferase (DNA MTase) family of enzymes catalyze the transfer of a methyl group to DNA. DNA methylation serves a wide variety of biological functions. All the known DNA methyltransferases use S-adenosyl methioni ne (SAM) as the methyl donor. In prokaryotes, the study role of DNA methylation is to protect multitude DNA against degradation by restriction enzymes. In eukaryotes, DNA methylation has been implicated in the control of several cellular processes, including ifferentiation, ingredient regulation, and embryonic development. morphologic work on HhaI DNA methyltransferase demonstrates that the substrate nucleotide is completely flipped out of the helix during the modification reception and has provided much insight into the enzymatic properties of S-adenosyl-L-methionine (SAM)-dependent DNA-modifying enzymes. Structural comparison of three enzymes, HhaI C5-cytosine methyltransferase, TaqI N6-adenine methyltransferase, and catechol O-methyltransferase, reveals a liaison similarity in protein folding and indicates that umpteen SAM-dependent methyltransferases have a common catalytic-domain structure.This mark permits the prediction of tertiary structure for some other DNA, RNA, protein, and small-molecule methyltransferases from their amino acid sequences, including the eukaryotic CpG methyltransferases. Ethidium cliche is an intercalating agent commonly used as a fluorescent tag (nucleic acid stain) in molecular biology laboratories for techniques much(prenominal) as agarose gel electrophoresis. It is commonly abbreviated as EtBr, which is also an abbreviation for bromoethane.When undecided to ultraviolet light, it will fluoresce with an orangeness colour, intensifying almost 20-fold after backbone to DNA. Ethidium bromide is an intercalating dye, that is, it is able to mooring line itself into the DNA while essentially stacking itself surrounded by the bases of the helix. When it is inserted into the DNA, it becomes much more fluorescent when exposed to ultraviolet light as compared to ethidium bromide just in solution. So we burn down use it to visualize the DNA that has been mulish on a gel by electrophoresis.