Lab 1: DNA Extraction
Materials:
¼ Kiwi fruit
2.5g washing up liquid
1g salt
50mL tap water
Ice cold alcohol; isopropanol（put in freezer for at least 30 mins）
Tools:
Knife to cut a kiwi in to ¼
Zip-lock bag for mashing a kiwi in
A pair of scissors for chopping
Major
Pen to label mu name on the bag
A small case to put the materials of extraction buffer and mix them in
Beaker
Centrifuge
Filter paper
Funnel and funnel stand
Bunsen burner
A long glass hook
Clean glass test tube
pH paper
Plastic micro-centrifuge tube
Blue pipetman
Procedure:
- Peel the kiwi fruit into 1/4 and chop it into small chunks using scissors.
- Put the chunks in a zip-lock bag and mash the kiwi to break up some of the cells and provide a large surface e area over which to extract the DNA.
- Make the extraction buffer by mixing together the washing up liquid, the salt and the tap water. Stir slowly until the salt has dissolved, not making bubbles.
- Add the extraction buffer to the mashed up kiwi and mash more to get out DNA at the end.
- Label the bag with my name and incubate the kiwi and extraction buffer mixture at 60 Celsius for 15 min in the water baths. Incubation helps to break up the cells further and starts to degrade some of the cell’s proteins. This is because Kiwi, like some other tropical fruits have special enzymes called proteases which are proteins which cut up or digest other proteins.
- Remove my bag from the water bath and filter the kiwi mixture into a beaker using filter paper, a funnel and a funnel stand. Filtration removes all the unwanted lumps and bits of kiwi fruit ad should leave you with a salty and soapy green liquid: extract which contains the kiwi fruit DNA.
- Pour some of this extract into a large glass tube and carefully add an equal volume of cold isopropanol from a tube in the freezer by poring it slowly down the side of the jar. The alcohol forms a layer on top of the kiwi extract because it has a lower density. Between the two layers of liquid, there is a white jelly-like substance forming, this is DNA. Then, mix the liquids to make your DNA stick together.
- Make a long glass hook by carefully heating a glass Pasteur pipette in the flame of the Bunsen burner. Then, reach down this hook inside and gently fish out some of the kiwi DNA which is very long and fragile.
- One way to show that what you have isolated is DNA, is by checking its pH when dissolved in water.
- Transfer thee fished DNA into a labeled plastic micro-centrifuge tub and pour in some of your unfinished liquid until the rube is almost full. Centrifuging this material for 2 minutes to cause the heavy aggregates of DNA to form a “pellet” in the bottom of the tube. When I put it in the centrifuge, I should balance the tube with another off the same volume to prevent that the centrifuge doesn’t suffer.
- Remove the liquid above the pellet using a pipetman and a blue tip.
- Resuspend the pellet using 1mL of distilled water and a new blue tip.
- Check and record the pH off the DNA solution using Hp paper. Also measure the pH of an appropriate “control”.
Results: Until before I incubate the kiwi and extraction buffer mixture at 60 Celsius for 15 min in the water baths, I couldn’t see any specific changes in the zip-lock bag. After the incubation, I could see the color change. It was light green before but it became yellowish green after the incubation. Therefore after I filtered the kiwi and extraction buffer mixture, I got a clear yellow liquid as I showed below.
When I added an equal volume of cold isopropanol into the tube which the filtered the kiwi and extraction buffer mixture in, I quickly could see some changes in it. As I wrote above in the procedure section, the alcohol forms a layer on top of the kiwi extract. Between the two layers of liquid, there is a white jelly-like and tiny bubble-like substance forming,
When I fished the DNA using the glass hook, I could feel that the DNA is really like a jelly, sticky and very condensed. After I centrifuged the DNA, it precipitated on the bottom of the micro-centrifuge tube and formed “pellet” there. It didn’t move even though I shook it hard.
After I poured the distilled water in the micro-centrifuge tube, the pellet was dissolved. Then, I measured its pH, it was pH 4, means it acid. I also measured the pH of an appropriate control which is the distilled water, it was neutral. Therefore, I can say DNA is acid.
Conclusions: I could actually take DNA out from foods through this process, could see it with my own eyes clearly and understand that it exists in all cells even though it is plant or food. According to the name of DNA which stands for Deoxyribonucleic acid, it should be acid, in this experiment, I could proof that DNA is acid indeed.
Comments/ Discussion: I could see some differences between my result and some other classmates’ one after the filtering and also after adding the isopropanol. I got clear yellowish liquid after filtering but some others got milky yellowish liquid on the other hand. That was maybe why they couldn’t get the jelly-like DNA. I wonder where these differences came from.
When I measure the extraction buffer by mixing together the washing up liquid, the salt and the tap water, I measured them very carefully and I followed the instruction step by step. Did this make some differences?
After we take the DNA out, is there any ways to see the famous spiral form of DNA using the normal microscope somehow? Since I know that as DNA is so thin, I wouldn’t be able to see it without an incredibly powerful microscope usually, so I wonder if I can see it or not.
Answer to Questions:
1), why did we use kiwi as our sample today?
To see DNA with my own eyes and understand that it exists in all cells even though it is plant or food. And also kiwi is soft to mash, and it is a fruit which we all are familiar with.
2). what was the washing up liquid for?
It dissolves the fatty cell membranes.
3). what did you use for your pH measurement control?
I used the same distilled water which I put into the plastic micro-centrifuge tube with pellet in.
4). what happens to the DNA that you eat and does eating modified DNA pose any danger to you?
GM foods are produced by artificially introducing new genes (DNA) into the cells of the organism to be modified. This is often done by a process called transformation in which "naked" DNA is added to cells and the DNA is assimilated by the transformed cells and incorporated into their chromosomes.
Eating modified DNA would pose some dangers to us. Manipulating genes is still under the research and even scientists are not crystal clear about its consequences. It is said that it might generate some unknown allergen or unexpected toxins. Therefore eating the genetically modified food is risky.
References:
http://www.newton.dep.anl.gov/askasci/mole00/mole00606.htm
http://www.smalltown.ne.jp/~usata/memo/gmo.shtml#food
http://www.seedsofdeception.com/GMFree/GMODangers/DangersofGMFoods/index.cfm
5). What does the term GMO mean?
It means the genetically-modified organisms.
References: http://www.raw-wisdom.com/50harmful.
http://www.newscientist.com/article/dn9921-instant-expert-gm-organisms.html
http://www.ornl.gov/sci/techresources/Human_Genome/elsi/gmfood.shtml
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