With the execution of this experiment, we began to go deeper into the Cell and Molecular Biology course. The main focus of the experiment would be how the Restriction Endonucleases cleave the strands of DNA. For this experiment, pBR322 was the specimen to use. Restriction Endonucleases work by cleaving the sugar phosphate backbone of specific DNA sites. Restriction enzymes that have been isolated from bacteria have a defensive role. This idea is illustrated when an attacking foreign cell DNA is trying to alter the bacteria; restriction enzymes cleave the DNA rendering it inert.
The second part of the experiment deals with Gel Electrophoresis. The samples are loaded into wells on an 1% agarose slab and subjected to electrical currents both positive and negative. Our current target here is DNA, therefore since nucleic acid as a negative charge, the bands will migrate toward the positive cathode. This process of migration is called sieving and smaller strands move faster than longer strands due to their ease in going through the gel.
The objectives of the experiment include:
Learning the principles behind Restriction Enzymes and Gel Electrophoresis Applying the concepts in the experiment to produce bands at the end of the Gel Electrophoresis stage Interpreting what these bands mean with accordance to how the plasmid was cleaved
Methods and Materials:
For the experiment we used several restriction 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 used 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 µL sterile distilled H2O, 1.0µL of the appropriate 10x buffer, 1.0µL combination of the restriction endonucleases and 1.0µL of pBR322 plasmid DNA (the DNA would be added last) in 5...