Today, I'm going to present a method of a very exciting experiment. It strongly ties in with what I have been discussing over the past week: DNA. Doing a biochemistry degree involves a lot of experimentation and this experiment is what I hope to be doing frequently during my education :)
The method of isolating DNA that I will present is the same across many parts of the world and is one of the most essential experiments in molecular biology.
Alright, gimme the main jist of it...
Okay, first we need to get an organism. I used an onion. Yes, it was a teary experiment. :')
We break up the tissue and use detergent to break down the cell surface membranes of... the cells and the nuclear membranes of the... nucleus. This is homogenisation.
The cell fragments are separated through filtration and we are left with DNA and soluble proteins.
To remove the protein, we use enzymes. Protease is a wise choice.
Finally, the DNA is precipitated using ethanol at around 250K (I personally kept it at 273K).
Apparatus
Liquidiser (mixer)
Knife and chopping board to cut onions
Water both that is ice cold
Thermometer
Funnel
Filter paper
500cm^3 Beaker
Glass rod
Stopwatch
Boiling tube and rack
25^3 measuring cylinder
Pipette
Sodium chloride solution (I used 4%M)
Low temperature ethanol (I used 95%M)
Detailed method please!
1. Slice onion with a knife into 5mm cubes and place in beaker with 100cm^3 detergent.
2. Stir and keep at 333K in a water bath for 15 minutes. The temperature breaks down the cell membranes of the onion and the detergent forms complexes surrounding the membrane phospholipids and proteins causing them to solidify out of the colution. Also, the Na+ sodium ions shield the negatively charged phosphate groups of DNA molecules causing them to coalesce.
(By this point, you will be crying. I shall give you the biochemical reason for this next time!)
3. Cool the mixture in iced water for 5 minutes whilst stirring. This slows down any breakdown of DNA if you kept it in high temperatures any longer.
4. Liquidise the mixture for ONLY 5 seconds - yeah it's that quick. This degrades the cell walls and membranes which allows more DNA to be released. The reason why liquidisation should be quick is because this stops the DNA strands from being completely and utterly broken up, which kinda destroys the experiment...
5. Filter it into a measuring cylinder and the filtrate should contain the DNA and soluble proteins.
6. Store this in a fridge for around 1 or 2 days
7. Add 1cm^3 enzyme to 20cm^3 of the onion tissue extract in a boiling tube and mix. The enzyme will hydrolyse the proteins associated with DNA.
8. Induce a layer of ice-cold ethanol on top of the mixture by pouring it slowly down the SIDE of the boiling tube.
9. Leave the tube for 3 minutes. The DNA is insoluble in ice-cold ethanol. Bubbles may form and the DNA will precipitate out.
10. Gently rotate the glass rod in the liquid at the interface of the alchol/detergent mix. The interface is a flash term for the point at which the two layers meet. Be careful not to stir too much as the mixing of layers would break up the fragile strands of DNA ~
11. A white web of DNA (which would look a lot like mucus) can be viewed. It can be drawn from the tube with a pipette and suspended again in a solution of sodium chloride or distilled water.
I hope this experiment helps people who may be getting ready to do their EMPAs or ISAs in the UK. This will help anyone who has a more practical approach to this very practical science of Biochemistry. Next week, inspired by my crying during when I carried out this experiment, I'm going to explain why we cry when we chop onions (and maybe a few old-wives tales too!).
Exocytosis
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