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ottimo lavoro, pochi errori lievi, mancano alcuni post.

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Purpose: To separate DNA fragments with gel electrophoresis  and compare them to discover the culprit.

Laboratory equipment:

micropipette

tips

tubes

gel tray

comb

sponge rack

incubator

centrifuge

graduated cylinder

conic flask

TBE solution

agarose

DNA

restriction enzyme

dye

electrophoresis

UV lamp

Method:

the first thing to do is prepare the agarose gel. Put in a conic flask 8 gr of agarose, TAE solution diluted and B-green gel dye, then mix and put in microwave for few minutes. Cover the container with plug to prevent the solution from splashing out but don't close it very tight. During heating mix the solution and pay attention it is full dissolved and not only partially. Now put the solution in a gel tray with a comb to obtain wells where put DNA samples. Remove any bubbles with a tips and wait 20 minutes to solidify.

In the meantime, cut DNA with restriction enzyme. Restriction enzyme cut DNA in particular sites and make fragments of different sizes: they could be short, medium or long. Transfer 3 ul of DNA from stock micro-tubes called C(culprit) and 1-2-3(samples) to your own corresponding ones. Add 17 ul of restriction enzyme to each micro-tubes and mix well. Put the tubes into the centrifuge for 30 seconds at 8000 rpm.  Move the tubes into the sponge rack and put them into the incubator for 30 minutes. After that, add 4 ul of loading dye to each tube.

Prepare the gel electrophoresis: take the gel consolidated, remove the comb and put in the gel tank, fill with buffer solution diluted.

Electrophoresis:  load 20 ul of each sample into 4 wells of the gel respecting the sequence C-1-2-3-4. Close the lid of the chamber and insert the plug in the power supply. Set the power supply to 90 volt for 35 minutes and start electrophoresis. The electrophoresis principle is based on different speed rates of different size fragments towards anode and the two forces that control the speed of fragments are electrical power and frictional force. DNA fragments run from the negative pole to the positive because they have a negative charge. The agarose gel is like a net, the shortest fragments will run quickly instead the longest ones will be slowed down and we can see them nearer the well. Shorter DNA fragments travel through the gel faster than a sample with larger fragments.

Compare the samples: Stop the electrophoresis and unplug the power supply, transfer the gel onto a transilluminator and turn it on. You should compare the sample with the DNA of the scene and discover the culprit.

Observation: DNA finger printing was used since 1984. It was developed by Alec. J.Jeffreys,  a molecular geneticist at the University of Leicester, Uk.  It was used for the first time in 1986 during a case of  two teenage girls's  murderer in England.  Nowadays this technical is very improved, it allows to compare billions or trillions DNA's profile and it is enough less than an anagram of DNA to identify a person. DNA finger printing is used to identify people but also for paternity test. At the end of the experiment we could identify the culprit comparing the DNA fragments of the samples with DNA of the crime scene. During the experiment you must be pay attention using DNA dye because it is dangerous for human health, it has carcinogenic effects.

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What role does DNA evidence play in solving crimes?

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What role does DNA evidence play in solving crimes?

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Why has DNA received a  lot of media attention?

DNA has received a lot of media attention because it has a fantastic power of discrimination. It is a strong evidence more than eyewitness and it allows to compare billions or trillions DNA's profile.

How long has DNA  been used to identify people?

DNA has been used to identify people since mid-eighties but during the years the technical is improved.

The techniques used nowadays are more sensitive. How much DNA is needed to do a test?

Nowadays is enough less than an anagram (WW nanogram) of DNA instead 50  nanograms or a microgram of DNA in the past.

How many cells are needed to get a full profile?

Three or four cells of the property are needed to get a full profile.

In what conditions must the exhibit be kept?

The exhibit be kept in bone dry conditions and if it's possible you can freeze it in a piece of  tissue.

How long does DNA last if taken in good conditions?

DNA taken in good conditions will last essentially forever.

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What is the basic principle of gel electrophoresis?

The electrophoresis principle is based on different speed rates of different size fragments towards anode. As agarose gel is like a filter, the shortest fragments will run quickly instead the longest ones will be slowed down and we can see them nearer the well. Shorter DNA fragments travel through the gel faster than a sample with larger fragments.

Why does DNA migrate toward positive electrode?

DNA migrates toward positive electrode because it has a negative charge.

What is the TBE composition?

The experimental conditions for digestion are defined at 37°C, pH controlled by TBE buffer solution, presence of Mg ions, Na Cl for the proper ionic strength (i).

Why is important to add Mg ions? Are they co-enzymes or cofactors?

Add Mg ions is important to focus activity on enzymes. They are co-enzymes.

How can DNA be detected in agarose gel?

Gel Electrophoresis the gel sheet is laying inside the gel tray. There are wells where DNA extracts are carefully loaded. TBE solution soaked into the gel. Applying the right voltage negatively charged DNA fragments will travel through the gel sheet towards positively charged electrode.

Why are DNA dying dangerous for human health?

DNA dye are dangerous for human health because it can produce carcinogenic effects.

What should be done to prepare an agarose gel?

At first, mix up agarose powder with buffer solution. Then heat the mixture in microwave a wide container. Mix solution during heating to full dissolve the agarose. When we preparing agarose is should be better wear gloves because some substances could be dangerous.

What should not be done preparing an agarose gel?

Over the container the cover must not be loose, this is to prevent the solution from splashing out. The solutions should not be partially dissolve but should be full dissolved otherwise the result will not be accurate.

Which piece of information can be find out from a DNA pattern bands?

We will see a fragment pattern: the sample with the same pattern found in the crime scene is the culprit.

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Was it possible to identify people in the past?

Before DNA technique discovery it was possible to identify people in other different ways using finger-printing since 1901, other observations led to find different types of particular proteins that could be present or absent.
How?  Evidence were compared with suspect 's pattern but the result of this investigation could be only used to exclude from a crime and not to accuse anybody.

When was used first DNA finger printing?

DNA finger printing was used since 1984. It was developed by Alec. J.Jeffreys,  a molecular geneticist at the University of Leicester, Uk.  It was used for the first time in 1986 during a case of  two teenage girls's  murderer in England.

Where can DNA be obtained from?

DNA can be obtained from biological materials like blood, semen, hair with roots, skin, dandruff, sweat stains, vaginal fluids, nasal secretions, urine, feces.

Who invented RFLP technique? When?

RFLP ( Restriction Fragments Lenghts Polymorphism ) technique was invented by Alec J. Jeffreys in 1984.

What is a restriction enzyme?

Their action mechanism provides to cut in pieces double strand viral DNA injected by viral particles. They are classified as endonucleases, we know about three thousand different types of restriction enzymes.

What is restriction enzyme role in nature?

Restriction enzymes have been developed inside bacteria cytoplasm to prevent viral o bacteriophage infection.