21 May 2011

Introduction to DNA Extractions

Lana Hays
Access Excellence Fellow
Simon Kenton H. S.
11132 Madison Pike
Independence, KY 41051

Notes to the teacher:
Through two weeks of numerous trials, I have learned a great deal about DNA extractions. I originally worked with eight different protocols to modify, simplify, and develop good extraction labs. My intent was to produce simple DNA extractions that use various types of cells. The materials used come from the grocery store, health food stores, and butcher shops. Several extractions require a centrifuge. I made it a point to get a centrifuge this year so that I could run the experiments. If you do not have access to a centrifuge, you might run the extractions as written and try to create ways to get around it. The centrifuge step can be skipped in the thymus experiment and good results still obtained. It's amazing what steps can be eliminated or modified.

It is best to begin collecting the materials two weeks in advance. All materials that you extract from must as fresh as possible. The two most difficult items to obtain are non-roasted wheat germ and calf thymus. Health food stores usually carry the non-roasted type of wheat germ, as do some large grocery stores. Thymus (sweetbread) will need to be ordered from a butcher shop. As butcher shops don't always know what will be slaughtered ahead of time, I had several shops trying to get it. Liver is not difficult to get but should be ordered fresh. Once purchased, thymus can be frozen until you need them. Cut them into chunks before freezing so that you can get just what you need each day. The other items can be purchased at most any large grocery store.

I like for my students to do as much of the protocol as they can. With several blenders and a little organization, all the labs can be completed in a 55 minute period or less. Each protocol produces enough lysate for 10-15 spoolings. If you are short on time and equipment, you can demonstrate the first part and then have the students complete the rest of the lab. If you want the individual students to complete all of the protocol, cut the quantities down proportionatey until you get to the spooling step. At this point, use the original protocol. The blender can handle mixing 10-15 ml of solution but you may need to cut the blending time down.

When DNA extractions are performed, you can expect three basic results.

    No DNA
    DNA appears fluffy which means it has sheared in the extraction process
    DNA appears as thin threads.

Although DNA that strands is the most impressive, DNA that has sheared still shows that DNA is present.

All the experiments will yield DNA but some more than others. The lima bean bacteria, and yeast give the poorest results. I'm sure that with some more experimentation they could be improved greatly. The most impressive is the calf thymus, so I have students do it last. The long threads of DNA are easily spooled and the quantity is immense compared to the other extractions.

If you have students do many DNA extractions, you will find that their lab skills will improve. However, a problem that constantly persists occurs when they add the alcohol--the students usually pour it too fast. Rather than forming two distinct layers, they mix the two. Once that happens, there's not much that can be done.

The experiments work well as written. However the following substitutions can be used:

    A reusable coffee filter or cheese cloth can be used to strain the materials

    91-99% isopropyl (rubbing alcohol) can be substituted for ethanol,although I prefer ethanol

    Fresh papaya or pineapple juice can be substituted for the meat tenderizer solution (use the same amount of ml as the meat tenderizer solution)

    10% SDS (sodium dodecyl sulfate) can be used in place of all the detergent solutions. It comes as a 10% solution already mixed or you can buy the powder and mix a 10% solution (5g SDS and 50ml distilled water).

A good science project for students is to run through several of the protocols. Then have them design modifications to test. They can use the substitutions, use other things to extract from, or switch solutions/protocols. You can have the students pipet the alcohol/DNA layer off and place it in a clean test tube to view later. At the end, they can compare the DNA from all the extractions or even create other labs in which to use the DNA. An excellent experiment is to have students run all the DNA extractions except yeast. Have them analyze the other extractions using the summary chart and research the characteristics of yeast. Based on their findings, have them design a protocol for yeast DNA extraction, run the experiment, and justify their results.

There are two to three basic steps in DNA extraction. The cell must be lysed (broken open) to release the nucleus. The nucleus (if present) must also be opened to release the DNA. At this point the DNA must be protected from enzymes that will degrade it, causing shearing. Once the DNA is released, it must then be precipitated in alcohol.

In order for the cell to be lysed, the lipid walls must be broken down. The detergent and salt solutions accomplish this. Cell walls, cell membranes, and nuclear membranes are also broken down by the action of the blender. In all but one protocol I eliminated the use of heat. Some references state that a temperature of 60oC is necessary to denature the DNAase enzymes that cause shearing in DNA while DNA is denatured about 80oC. Other references state that DNA can denature at 60oC. From all the experiments I ran (except for the wheat germ protocol), I had sheared DNA when I used heat. Heat may destroy the enzymes as well as the DNA. However keeping the solutions cool seems to slow the enzyme action. The prep solution uses epsom salts and buffered aspirin to further deactivate the enzymes that degrade DNA when released and stabilize the DNA (acid vs. Base). Sodium bicarbonate (baking soda) also is used to buffer the solution. The meat tenderizer has papain, an enzyme that helps clean the protein from the DNA that can contaminate it. Papaya juice and pineapple juice also contains this enzyme. Finally, the ethanol is used to precipitate the DNA. In water, DNA is soluble. When it is in ethanol, it uncoils and precipitates leaving behind the other cell components that are not soluble in ethanol.

All in all, the DNA extraction labs are very workable. Try some and then decide if you would like to modify any to fit your needs better. Good luck!!

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