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DNA Extraction and Experiments on bacteria
Julian Lui, Dr. Y.K. Suen, Erika Ngan & Rebecca Lee
19th Nov, 2005
What is DNA?
Deoxyribonucleic acid (DNA) Building units of genetic materials Gene: a region of DNA that controls a
hereditary characteristics Chromosome: a group of coiled
strands of DNA, containing genes 23 pairs of chromosomes
Discovery of DNA
Rosalind Franklin's X-ray diffraction photograph of DNA
http://www.npr.org/programs/atc/features/2002/oct/darklady/
Components of DNA
Basic unit: nucleotide Nucleotide: deoxyribose + nitrogen co
ntaining base + phosphate group
http://faculty.uca.edu/~benw/biol1400/notes13.htm
DNA backbone
Form bonds between
sugar and phosphate
(sugar-phosphate
backbone)
http://fajerpc.magnet.fsu.edu/Education/2010/Lectures/24_dna.htm
Function of DNA
The Central Dogma:
DNA
RNA
Protein
Transcription:The synthesis of anRNA copy of a Segment of DNA
Translation
Hereditary diseases (1):
http://www.lwh.org.uk/clinical_services/genetics/cytogenetics/about_us.html
Down’s Syndrome
Hereditary diseases (2):
Thalassemia Inherited disease of red blood cells Defect in RBC formation, leading to severe ane
mia
http://en.wikipedia.org/wiki/Thalassemia
Outline of experimental work today
Chromosomal DNA isolation from Onion/Pig Liver/Kiwi
Bacterial clone isolation from Yakult
Chromosomal DNA isolation (1)
(for pig liver, cut the liver into small pieces with scissors after addition of detergent and NaCl)
50ml 10% detergent + 1 spoonful of NaCl, stir
Incubate at 50-60oCfor 10min
Crush the onion/kiwi with a mortor
Transfer the liquidportion with a dropper
Add 1-2 drops of meat tenderizer solution & mix
Chromosomal DNA isolation (2)
Add equal volume of ice cold ethanol
Leave at room temp. & wait
Precipitated DNA strand
Dry with papertowel
White fiber-like precipitate
DNA detection
DNA detection
Onion DNADNA marker
Dry the precipitate with paper towel
Place the precipitateon saran wrap under a UV light source, add one drop of EtBr
Resuspend the DNA with 50l0.5M NaCl, run 20l DNA on 1%agarose gel
DNA stained with EtBr
Observe under money detector
Uses of reagents
Detergent Permeabilizes cell wall/ cell membrane to rel
ease cellular contents NaCl
Improves dissociation of DNA from other cellular components
Meat tenderizer Contains enzymes which help to digest prote
ins in the cell lysate Ethanol
Precipitation of DNA
Ethidium Bromide
Formula: 2,7,-Diamino-10-ethyl-9-phenyl-phenanthridium bromide
Non-radioactive marker for identifying and visualizing nucleic acid bands in electrophoresis
Emits reddish-brown fluorescence when bound to DNA or RNA and exposed to UV light
Hazards: EB is a potent mutagen and moderately
toxic and carcinogenic (may cause cancer)
EB can be absorbed through skin, so it is important to avoid any direct contact with the chemical
Different methods for DNA quantification By staining DNA with dyes
Quick detection (by UV light source)• SYTO16• Methylene blue• Propidium iodide
Visualization on agarose gel• Ethidium bromide
Measuring absorbance (OD) at 260nm
What is inside Yakult?
Lactobacillus casei Shirota strain Naturally occurring bacteria found in small i
ntestine Highly acid resistant Beneficial effects:
- promote bowel health- increase the no. of beneficial bacteria in the large intestine, e.g. Bidifidobacteria
1ml of Yakult contains 100 million L.Casei Shirota strain bacteria
http://www.yakult.com.au/education02.htm
Major activities of the lactic acid bacteria
Promote health
Diminish the no. of detrimental bacteria in the digestive systemRegulate bowel movement by stimulating peristalsis, and help digestion and absorption
Provide the taste and flavour
The sour taste of fermented foods is due to lactic acid produced by the bacteria
Preservation of food
Acids in food help to prevent growth of pathogenic bacteria during storage
http://www.yakult.com.au/education02.htm
Preparation of agar plate
3.5g LB agar powder in 100ml distilled water
100ml LB agar solution
Sterilize the LB agar for 20min
Cool down to ~60oC
~20ml LB agar
Cool and solidify theagar at room temp.for at least 1 hr
Invert the agar plate and keep at room temp.overnight
Store at 4oC for future use
Culturing of Yakult
Warm and dry the LB agar plate in 37oC incubator
Sterilize the bench with70% ethanol and lit analcohol lamp
Add 3 drops of Yakult
Spread the droplets thoroughly on theagar plate using the spreader and incubatethe plate at 37oC overnight
Sterilize a glass spreader by soaking into 70% ethanol and briefly flame it with alcohol lamp
Other experiments utilizing LB agar plates (1)
1 2
4 3
Experiment on hand washing with different cleansing agents
Press thumb on quadrant 1
Wash with soap for 15secs
Wash with soap for another 15secs
Press thumb on quadrant 2
Press thumb on quadrant 3
Wash with soap for another 15secs
Press thumb on quadrant 4
Observe the no. of colonies at different quadrants and compare the results using different agents (water, soap and alcohol)