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/

Location of DNA

http://staff.washington.edu/dehlert/pages/clr_kids_celldna.html

Components of DNA

Basic unit: nucleotide Nucleotide: deoxyribose + nitrogen co

ntaining base + phosphate group

http://faculty.uca.edu/~benw/biol1400/notes13.htm

4 types of bases

Purine bases

http://www.galaxynet.com/~corvid/che/cher_bio_nucleic.htm

4 types of bases

Pyrimidine bases

http://www.galaxynet.com/~corvid/che/cher_bio_nucleic.htm

DNA backbone

Form bonds between

sugar and phosphate

(sugar-phosphate

backbone)

http://fajerpc.magnet.fsu.edu/Education/2010/Lectures/24_dna.htm

DNA double helix

http://bh.kyungpook.ac.kr/~sdsong/images/HB1-DNA%20DHelix.gif

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 from Onion/Pig liver/Kiwi

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

Bacterial Clone Isolation from Yakult

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)

Q & A

End of Workshop

Thank you!!