LAB3.docx - NAME Varsha Sooklal ID 66280 LAB 3 TITLE DNA ISOLATION FROM CHEEK CELLS INTRODUCTION The process of isolating DNA from a cell is the first

LAB3.docx - NAME Varsha Sooklal ID 66280 LAB 3 TITLE DNA...

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NAME: Varsha Sooklal ID: 66280 LAB# 3 TITLE: DNA ISOLATION FROM CHEEK CELLS INTRODUCTION: The process of isolating DNA from a cell is the first step for many laboratory procedures in biotechnology. The scientist must be able to separate the DNA from the unwanted substances of the cell gently enough so that the DNA is not broken up or shredded. The procedure done in this experiment is a comparison of DNA purity and quality using two types of DNA isolation kits for buccal cavity cells. Most DNA isolation procedures are modification of the “Marmur” preparation which is used worldwide in biotechnology laboratories. This is where a “Filtrate” is made of live tissue treated with salt, distilled water and detergent (SDS). The tissues that are used because it has a low starch content which allows the DNA to be seen more clearly. The salt shields the negative phosphate end of DNA which allows these ends to come closer so they can precipitate out of a cold alcohol solution. The detergent causes the cell membrane to breakdown by emulsifying the lipids and proteins of the cell and disrupting the polar interactions that hold the cell membrane together. The detergent then forms complexes with these lipids and proteins, causing them to precipitate out of solution. Collectively, the salt solution and detergent are referred to as a lysing buffer. AIM: To determine the purity of DNA from buccal cells. METHODOLOGY Materials/Equipment: Isohelix kit.
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PROCEDURE: BEFORE STARTING 1. A waterbath at 60 0 C was prepared. 2. No precipitate was formed in the LS solution, and was then warmed at 60 0 C for a few minutes. 3. The PK solution from the freezer and was allowed to thaw at room temperature. ISOHELIX DDK SWABS Isohelix DDK swabs are intended for the retrieval of buccal cells, for single use only. The swabs are kept at room temperature. The package received consisted of fresh silica gel which is coloured orange and only changes green when exposed to moisture. 1. At least 1 hour after eating and rinsing of the mouth with water before the DNA samples were taken. 2. The package was opened and the swab was removed, making sure that the swab did not touch the fingers. 3. The swab was then inserted into the mouth and rubbed firmly against the inside of the cheek for 1 minute and was placed back into the tube carefully. 4. The silica gel capsule was then added to the tube for about 1 minute and was then removed and disposed leaving only the swab in the tube and the tube was sealed. DNA ISOLATION PROTOCOL Part A – DNA Stabilization 1. 500µl of the LS solution was added to the tube containing the buccal swab. 2. 20µl of the PK solution was then added to the tube containing the buccal swab and the LS solution and was vortex briefly.
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