Biotech Basics Fundamentals of Electrophoresis Danielle R Snowflack
Biotech Basics: Fundamentals of Electrophoresis Danielle R. Snowflack, Ph. D. www. edvotek. com © EDVOTEK, THE BIOTECHNOLOGY EDUCATION COMPANY - WWW. EDVOTEK. COM - 1. 800. EDVOTEK
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Today’s Experiment: Agarose Gel Electrophoresis • Agarose gel electrophoresis rapidly separates molecules into discrete bands based upon charge, size, and shape. • Principles and Practice of Agarose Gel Electrophoresis (Edvo. Kit 101) • Features Ready-to-Load™ Dye Samples © EDVOTEK, THE BIOTECHNOLOGY EDUCATION COMPANY - WWW. EDVOTEK. COM - 1. 800. EDVOTEK
What Do I Need to Perform Electrophoresis Experiments? • Samples: dye, protein, RNA, DNA • Agarose • Electrophoresis Buffer • Electrophoresis Apparatus • D. C. power source • Micropipet © EDVOTEK, THE BIOTECHNOLOGY EDUCATION COMPANY - WWW. EDVOTEK. COM - 1. 800. EDVOTEK
Overview of Electrophoresis © EDVOTEK, THE BIOTECHNOLOGY EDUCATION COMPANY - WWW. EDVOTEK. COM - 1. 800. EDVOTEK
Casting the Agarose Gel https: //youtu. be/EZj. Nuq. SEPb. Y © EDVOTEK, THE BIOTECHNOLOGY EDUCATION COMPANY - WWW. EDVOTEK. COM - 1. 800. EDVOTEK
Preparing for Electrophoresis https: //youtu. be/lgmq_Hsu. ZIU © EDVOTEK, THE BIOTECHNOLOGY EDUCATION COMPANY - WWW. EDVOTEK. COM - 1. 800. EDVOTEK
Calibrated Micropipettes Measure Small Volumes 1. CHOOSE the correct micropipet for the volume you are measuring. 2. SET the volume by twisting the top of the plunger. © EDVOTEK, THE BIOTECHNOLOGY EDUCATION COMPANY - WWW. EDVOTEK. COM - 1. 800. EDVOTEK
Measuring Liquids with a Micropipet 1. 2. 3. 4. 5. 6. SET the micropipet to the appropriate volume. PLACE a clean tip on the micropipet. PRESS the plunger down to the first stop. HOLD the plunger down while placing the tip beneath the surface of the liquid. Slowly RELEASE the plunger to draw sample into the pipette tip. DELIVER the sample by slowly pressing the plunger to the first stop. DEPRESS the plunger to the second stop to expel any remaining sample. DO NOT RELEASE the plunger until the tip is out of the sample container. DISCARD the tip by pressing the ejector button. Use a new tip for the next sample. © EDVOTEK, THE BIOTECHNOLOGY EDUCATION COMPANY - WWW. EDVOTEK. COM - 1. 800. EDVOTEK
Let’s run our gel! • Variable Micropipette 20 -200 • Duo. Source 150 • Edvo. Kit 101 – Principles and Practice of Agarose Gel Electrophoresis • M 12 Complete Electrophoresis Package © EDVOTEK, THE BIOTECHNOLOGY EDUCATION COMPANY - WWW. EDVOTEK. COM - 1. 800. EDVOTEK
The History of Electrophoresis • In 1937, Arne Tiselius developed moving boundary electrophoresis. • A U-shaped tube is filled with a sample and buffer and then charge is applied to the ends of the apparatus. • Based on the charge of the molecules, they would move towards the positive electrode or the negative electrode. • Separation was visualized by the change in the refractive index at points along the tube. © EDVOTEK, THE BIOTECHNOLOGY EDUCATION COMPANY - WWW. EDVOTEK. COM - 1. 800. EDVOTEK
The History of Electrophoresis • Researchers experimented with different matrices (including paper and starch) to separate molecules into separate bands before settling on the use of agarose. • Today, electrophoresis is one of the most common biotechnology techniques used in the research laboratory. © EDVOTEK, THE BIOTECHNOLOGY EDUCATION COMPANY - WWW. EDVOTEK. COM - 1. 800. EDVOTEK
Electrophoresis Chambers for Classrooms of all Sizes Cat. # 502 Model M 12 Two gels Cat. # 515 Model M 36 Six gels © EDVOTEK, THE BIOTECHNOLOGY EDUCATION COMPANY - WWW. EDVOTEK. COM - 1. 800. EDVOTEK
Power Supplies Provide Current for Electrophoresis Cat. #509 Duo. Source™ 150 (75/150 V) Cat. #5010 Quadra. Source™ (10 -300 V) © EDVOTEK, THE BIOTECHNOLOGY EDUCATION COMPANY - WWW. EDVOTEK. COM - 1. 800. EDVOTEK
Introducing the Edvotek EDGE™ © EDVOTEK, THE BIOTECHNOLOGY EDUCATION COMPANY - WWW. EDVOTEK. COM - 1. 800. EDVOTEK
Principles of Electrophoresis Dye molecules have an overall net charge which influences their movement through the gel. Crystal Violet positive Orange G negative Xylene Cyanol negative © EDVOTEK, THE BIOTECHNOLOGY EDUCATION COMPANY - WWW. EDVOTEK. COM - 1. 800. EDVOTEK
Electrophoresis Separates Dyes By Charge • The samples are loaded into the gel, and an electrical current is passed through the gel. • The current drives the negatively charged dyes through the gel towards the positive electrode, and positively charged dyes to the negative electrode. • The dyes separate into distinct zones based on their charge. © EDVOTEK, THE BIOTECHNOLOGY EDUCATION COMPANY - WWW. EDVOTEK. COM - 1. 800. EDVOTEK
Electrophoresis Separates DNA Fragments By Size Edvotek Kit 130 • The sugar-phosphate backbone of DNA has a strong negative charge. • The current drives the DNA fragments through the gel towards the positive electrode. • Small DNA fragments move through pores easily, but large DNA fragments have a more difficult time squeezing through the tunnels. • The DNA separates into discrete bands. © EDVOTEK, THE BIOTECHNOLOGY EDUCATION COMPANY - WWW. EDVOTEK. COM - 1. 800. EDVOTEK
Summary of Electrophoresis © EDVOTEK, THE BIOTECHNOLOGY EDUCATION COMPANY - WWW. EDVOTEK. COM - 1. 800. EDVOTEK
Results and Conclusions • Gel electrophoresis rapidly separates molecules based on their physical and chemical properties. © EDVOTEK, THE BIOTECHNOLOGY EDUCATION COMPANY - WWW. EDVOTEK. COM - 1. 800. EDVOTEK
Principles and Practice of Agarose Gel Electrophoresis • Agarose gel electrophoresis rapidly separates molecules into discrete bands based their physical and chemical properties. • Since its inception, electrophoresis has evolved into the powerful biotechnology technique we use today in labs worldwide. © EDVOTEK, THE BIOTECHNOLOGY EDUCATION COMPANY - WWW. EDVOTEK. COM - 1. 800. EDVOTEK
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