Introduction to Gel Electrophesis

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Title: Introduction to agrarose gel electrophoresis. Name: D Creaven Date: 4th February 2013 Module: Forensic DNA Laboratory Tutor: Dr Colin Conway Title Introduction to agrarose gel electrophoresis. Date 4th February 2013 Lab Partners Jurgita Baltrukoniene Janet McHugh Objective (a) To prepare the agrarose gel electrophoresis (b) To analysis the movement of DNA on the agrarose gel. Introduction Agarose gel electrophoresis is an easy way to separate DNA fragments by their sizes and visualize them. It is a common diagnostic procedure used in molecular biological labs. Electrophoresis: The technique of electrophoresis is based on the fact that DNA is negatively charged at neutral pH due to its phosphate backbone. For this reason, when an electrical potential is placed on the DNA it will move toward the positive pole. Materials Agarose (electrophoresis grade), 1 x TAE (0.4M Tris-acetate; 0.01M EDTA; pH 8.0), 6 x Gel loading dye, Ethidium bromide stock (10mg ml ⁻¹) Store at room temperature protected from light, DNA sample material (e.g 100 bp DNA MW ladder, lambda phage), Micropipettes and tips, Microcentrifuge tubes, 1.5 ml eppendorf tubes, Microfuge, Transilluminator Procedure 1. A gel mould was prepared by sealing the open ends of Perspex through with autoclave tape and a sample comb positioned 0.5-1.0 mm above the surface of the trough. 2. A 1% agrarose gel was prepared by dissolving 0.5 g agrarose in 50 ml of 1X TAE in an Erlenmeyer flask or glass bottle with a loose fitting. 3. This was allowed to cool to approximately 55ºC and ethidium bromide (10mg ml⁻¹) was added to a final concentration of 0.5 μg ml⁻¹. 4. The gel was poured to a depth of about 5 mm, taking care to avoid bubbles and was allowed to solidify for 30 mins at room temperature. 5. The tape was removed and the trough containing the

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