1-LabManual2011

1-LabManual2011 - Heredity Lab Manual DNA Extraction From...

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Heredity Lab Manual DNA Extraction From Onion Tissue Goal: To visualize a large quantity of DNA. Almost everyone knows what DNA is: the genetic information for all living things. But what does it look like? The purpose of this lab is to give you hands-on experience with DNA by isolating it from onion tissue. DNA extraction requires 1) breaking open cells and releasing their contents, 2) separating DNA from the other cell contents (mostly proteins and carbohydrates), and 3) precipitating the DNA in order to further purify it and so it can be seen. After precipitation, the DNA is visible as a white-colored, stringy mass. Note: all DNA looks the same, regardless of from what organism it is extracted. MATERIALS blender onion lysis buffer (for 900mL) 45 mL 1M Tris (pH 8.0) 45 mL 0.5M EDTA 54 mL 5M NaCl per table: ice bath per groups of three or four: 250 ml beaker cheese cloth (4 layers) about 20 g of onion 10% SDS per pair: 100-1000 uL micropipettor 50 mL tube glass pipette w/ hooked end 15 mL ice-cold 95% ethanol ( in ice bath ) PROCEDURE Acquire the items above that you’ll need per group and per person. Lysing the cells ( complete the following steps in groups of three or four ) 1. Add about 15 g of diced onion with 75 mL lysis buffer and blend for about 45 seconds. Pour the homogenate into a beaker. 2. Place cheesecloth over another beaker, forming a depression. Hold in place with a rubber band. 3. Pour onion homogenate onto cheesecloth and let it sit for a few moments to filter.
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Precipitating DNA ( complete the following steps individually ) 1. Transfer 10 mL of filtered onion homogenate to a 50 mL tube (labeled with your name). 2. Using a micropipetter, add 1 mL of 10% SDS. 3. Mix by swirling and place tube in the ice bath for 5 minutes. 4. Add 15 mL ice-cold ethanol and mix by holding the capped tube horizontally and slowly rocking it back and forth. Watch as the DNA precipitates out of solution and becomes visible. Continue rocking the tube for a couple of minutes. 5. Spool the DNA onto the hooked end of a glass pipette by twirling the pipette in the DNA precipitate. Since the purpose of this lab is simply to visualize DNA with the naked eye, we will not do any further manipulation of the DNA. We will do that in subsequent labs. Cleanup: Rinse off the glass pipettes and return them to their original beaker. Rinse out other glassware and hang them on the drying rack in the storeroom. Return the micro-pipetters and tip boxes to their proper locations. Wipe off your benchtop area.
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Manipulation of DNA: Part I Cutting with Restriction Enzymes Goal 1: Use restriction enzymes to manipulate DNA molecules so that the sizes of DNA fragments can be determined. Plasmids are small, circular DNA molecules commonly found inside bacteria.
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This note was uploaded on 01/17/2012 for the course BIOL 303 taught by Professor Pfau,r during the Fall '08 term at Tarleton.

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1-LabManual2011 - Heredity Lab Manual DNA Extraction From...

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