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DNA FINGERPRINTING

Purpose: discover the culprit after making agarose gel and after the comparisoningr  of DNA fragments.

Material and Tools:

-          Tips

-          Two micro pipette ( capacity: 1-10 and 10-100 µL)

-          Comb

-          Green gel dye plus

-          TAE (Tris Acetate EDTA) 10X

-          Microtubes with four different DNA sample

-          Microtubes with restriction enzyme P.St 1

-          Empty micro tubes

-          Balance

-          Gel tray

-          Graduated cylinder

-          Conic flask

-          Press

-          Transilluminator

-          Electrophoresis Chamber

-          Sponge

-          Thermal bath

-          Microwave

-          Centrifuge

-          Loading dye

-          Agarose powder

-          Buffer solution

-          Water

-          Rack

Process:

-          Add 17µL of restriction enzyme, Mg ions and the buffer solution to 3µL of bacterial DNA in 4 microtubes (called C, 1, 2, 3). [phases of digestion]

-          Put all microtubes into the centrifuge for 30 seconds at 8000 rpm.

-          After put them in thermal bath at temperature of 37 °C for 30 minutes.

-          Make agarose gel: catch draw 20 mL of TAE 10X, 0,8% of agarose, 200 mL of water and put them in a graduate cylinder.

-          Put the solution in microwave and pour it in the electrophoresis’s chamber

-          Load 4µL of loading dye, respecting the loading sequence after having removed them from the termal bath

-          Add 20 µL of gel tray in each of microtubes

-          Close the lid of the chamber.

-          Insert the plug in the power supply.

-          Set the power supply to 90 Volt for 35 minutes.

-          Start electrophoresis.

-          Stop the electrophoresis and unplug the power supply.

-          Transfer the gel on a transilluminator.

-          Turn the transilluminator on and it is possible to see the DNA fragments.

Observations:

It is possible to see that ‘1’ coincides with ‘C’, as a matter of fact ‘1’ and ‘C’ have the same disposition of DNA fragments.

Conclusion:

Through the use of the electrical current and the frictional force between gel and molecules, the gel electrophoresis he is able to separate different length DNA fragments, as a matter of fact the first force moves the molecules to the positive pole, while the second force breaks more the bigger one.

It is possible to see that the longer fragments run slower in the gel than the smaller.

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Ho inserito alcune correzioni. Nel complesso ti sei impegnata e hai effettuato un lavoro ricco di diverse parti, manca la relaz di laboratorio.
VF

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https://www.thinglink.com/scene/874709565839507458

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In my opinion is very important:
- voltage applied to the electrophoretic cells: in fact positive and negative pole must be applied in the correct way because DNA goes to the positive pole.
- buffer solution pH: hydrogen can influence DNA concentration
- agarose gel concentration and the DNA fragment lenghts: if the gel is too fluid or dense, DNA pass though net of agarose gel in a different ways.

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14"  Why has DNA received a  lot of media attention?
Because it has a fantastic power of discrimination.

36"   How long has DNA  been used to identify people?
Probably since mid-eighties.

52"   The techniques used nowadays are more sensitive. How much DNA is needed to do a test?
Less than a NANOgram of DNA.

1'26 "How many cells are needed to get a full profile?
Three of four cells.

1' 46" In what conditions must the exhibit be kept?
It's kept in damp conditions.

1' 52" How long does DNA last if taken in good conditions?
Essentially forever.

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1) Gel electrophoresis uses electricity to separate fragments of DNA based on their length.
2) The negative charge causes them to migrate towards the positive electrode when placed in an electric field.
3)TBE or Tris/Borate/EDTA, is a buffer solution containing a mixture of Tris base, boric acid and EDTA. TBE and TAE buffers are often used in electrophoresis.
4) Mg+2 is a co-factor and it helps when we adda it.
5) Agarose gel electrophUoresis (sp) is the most effective way of separating DNA fragments of varying sizes.
To separate DNA using agarose gel electrophoresis, the DNA is loaded into pre-cast wells in the gel and a current applied. The phosphate backbone of the DNA molecule is negatively charged, therefore when placed in an electric field, DNA fragments will migrate to the positively charged anode. Because DNA has a uniform mass/charge ratio, DNA molecules are separated by size within an agarose gel in a pattern such that the distance traveled is inversely proportional to the log of its molecular weight. The leading model for DNA movement through an agarose gel is "biased reptation", whereby the leading edge moves forward and pulls the rest of the molecule along. After separation, the DNA molecules can be visualized under uv light after staining with an appropriate dye.
6)The death (DYE )of the DNA ? is dangerous to human health, because the cells can't fight high doses of radiation, which damage DNA.
7) - Measure out 0.80 g of agarose powder.
- Mix the agarose with 100 mL of 1X TAE in a microwavable flask
- Cover the flask and microwave for 1-2 minutes
- Cool down agarose solution for 5 minutes
- Add a final concentration of 5 μg/mL Ethidium Bromide to the flask
- Puor (SP) solution into casting tray
- Place comb within the casting tray
- Let casting tray sit for 5 minutes at 4°C or 20/30 minutes at room temperature
- Add 6 μL of 6x loading buffer to each of the digest sample
- Set up gel electrophoresis unit
- Fill gel box with 1X TAE until completely covered
- Load molecular weight marker into one lane
- Load digest samples into gel
- Plug in ge unit and run gel at 100v for about 90 minutes
- Visualize DNA fragments with UV light
8)
9)

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1)What is the basic principle of gel electrophoresis?
2)Why does DNA migrate toward positive electrode?
3)What is the TBE composition?
4)Why is important to add Mg ions? Are they co-enzymes or cofactors?
5)How can DNA be detected in agarose gel?
6)Why are DNA dying dangerous for human health?
7)What should be done to prepare an agarose gel?
8)What should not be done preparing an agarose gel?
9)Which piece of information can be find out from a DNA pattern bands?

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1) Yes, it is possible.
2)  In the past human identification has been historically based on fingerprinting.
3) The first DNA finger printing was used for the first maternity test.
4) DNA can be obtained from semen, urine, vaginal fluids, nasal secretion, feces, blood stain, skin, dandruff, sweat stain and hair with roots.
5) The English Scientist Alec Jeffries invented RFLP, in 1984.
6) they are classified as endonucleases, we know about three thousand different types of restriction enzymes.
7) Restriction enzymes have been developed inside bacteria cytoplasm to prevent viral o bacteriophage infection: their action mechanism provides to cut in pieces double strand viral DNA injected by viral particles.
(https://images.sciencedaily.com/2015/09/150928123446_1_900x600.jpg Photography)

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1)Was is possible to identify people in the past?
2)How?
3)When was used first DNA finger printing?
4)Where can DNA be obtained from?
5)Who invented RFLP technique? When?
6)What is a restriction enzyme?
7)What is restriction enzyme role in nature?