DNA Separation By Electrophorosis

DNA, Deoxyribonucleic acid, is a double stranded, helical nucleic acid moleculewhich determines inherited structure of a protein. The “steps” are made ofbases: adenine, guanine, cytosine, and thymine. The sides are sugar andphosphate molecules. Restriction enzymes are enzymes that cut DNA at restrictionsites, leaving fragments blunt or sticky.

The restriction fragments areseparated using a technique called gel electrophoresis. DNA has a negativecharge so when an electrical charge is applied it makes DNA move to the positiveside. DNA is placed in agarose gel. Smaller fragments move faster. The purposeof this lab is to separate DNA fragments using gel electrophoresis. Hind IIIcuts AAGCTT between the two irst As.

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EcoRI cuts at GAATTC between the G andthe A. Hind III and EcoRI both make sticky ends. Results: Our results for thislab were EcoRI separated into five fragments.

Hind III separated into fourfragments. The control only had one fragment. (See chart A and figure 1-1 fordistances) Discussion: The purpose of this lab was to see how gelelectrophoresis separates DNA fragments. We used Hind III, EcoRI, and acontrolled enzyme. Some fragments were hard to see because of smearing. Thesewere the bigger fragments. Loading the DNA was difficult and if you werentcareful you could rupture the wells which ruined the lab. We, fortunately, didnot run into this problem.

Abstract: The purpose of this lab is to separate DNAfragments with gel electrophoresis using EcoRI and Hind III. Restriction enzymesare used to break up the DNA, then negatively charged DNA is placed in a gelcasting tray. Then it is placed into an electrophoresis chamber. An electricalfield is placed across the agarose gel which forces the fragments to move downthe gel. The amount of lines show how many fragments there is in the DNA. We hadfive fragments for EcoRI and six for Hind III.

The no enzyme had only onefragment. Procedures: We sealed the ends of a gel casting tray with masking tapeand inserted a comb into the slots. The tray was filled about 6mm high withagarose gel. It covered half the height of the comb. We waited ten minutes forthe gel to solidify. Then we placed the tray in a gel box and made sure that thecomb was at a negative (black) end. The box was filled with tris-borate-EDTAbuffer so it covered the entire surface of the gel.

The combs were removedwithout ripping the wells. The micro pipet was used to load the lambda EcoRI,lambda Hind III, and lambda only into the wells. We dipped the pipet trough thesurface of the buffer over the wells and expelled the contents. The top of theelectrophoresis chamber was closed and electrical leads were connected. The dyewas observed as it moved shortly after the power supply was turned on. The powersupply was turned off after the bands migrated near the end of the gel and thetop of the electrophoresis chamber was removed. We removed the gel from the gelcasting tray and examined it under a light box and compared it to the ideal gel(figure1-2).

BibliographyReferences: Restriction Enzymes: Cleavage of DNA lab University of Illinois.(1999). Experiment 2 Gel Electrophoresis of DNA.

In Molecular Biology Cyberlab,online: Http://www.life.uluc.edu/molbio/geldigest/electro.html