Practical Of Genetics

The Islamic University - غزة اإلسالمية الجامعة Faculty of Science العلوم كليةBiology Department األحياء قسم

Lab. 1

Laboratory practices and Plant DNA extraction at

home

• DNA : A nucleic acid that carries the genetic information in the cell and is capable of self-replication and synthesis of RNA.

• DNA consists of two long chains of nucleotides twisted into a double helix and joined by hydrogen bonds between the complementary bases adenine and thymine or cytosine and guanine.

• The sequence of nucleotides determines individual hereditary characteristics.

Some of Genetic Terms

• Genes : Segments of DNA that control cell structure and function.

Sequence of DNA bases usually code for a polypeptide sequence of amino acids.

• Allele: an alternate form of a gene. Usually there are two alleles for every gene, sometimes as many a three or four.– Any of the possible forms in which a gene for a specific

trait can occur. In almost all animal cells, two alleles for each gene are inherited, one from each parent.

• Paired alleles (one on each of two paired chromosomes) that are the same are called homozygous , and those that are different are called heterozygous.

• Dominant : a term applied to the trait (allele) that is expressed regardless of the second allele.

• Recessive : a term applied to a trait that is only expressed when the second allele is the same

• Phenotype : the physical expression of the allelic composition for the trait under study.

• Genotype: the allelic composition of an organism.

• Genome : The set of genes carried by an individual. – The entirety of an

organism's hereditary information. It is encoded either in DNA or, for many types of virus, in RNA.

– The genome includes both the genes and the non-coding sequences of the DNA/RNA.

• Chromosomes: Structures in the nucleus of an eukaryotic cell that consist of DNA molecules that contain the genes.

Genetics : The study of the structure and function of genes and the transmission of genes from parents to offspring.

• All of the chemicals listed below are highly toxic and hazardous compounds. Take extreme care when handling these compounds. On contact with skin/eyes wash immediately with water.

•  Ethanol (Ethyl Alcohol) :– Colourless liquid and highly flammable.– Irritating to eyes.– Intoxicating if ingested (If ingested in undiluted form,

it has a severe drying effect on mucous membranes of mouth and throat.) Wash out mouth thoroughly, and give plenty of water to drink. Do not induce vomiting.

• Methanol ( methyl alcohol ): – Colorless and flammable liquid. – Methanol is toxic. If ingested or inhaled it can cause a wide

range of harmful effects, from sickness, heart and liver damage to reproductive harm, blindness or death. Methanol can degrease the skin, which may cause dermatitis.

– In case of methanol contact with the skin, remove contaminated clothing, Wash with soap and water for 15 minutes.

– In case of methanol contact with the eyes, flush with gently running water for a minimum of 15 minutes. In case of inhalation of methanol vapours, remove the individual to fresh air. Asphyxiation from vapours may require artificial respiration.

• Acetic acid glacial: – Inhalation of concentrated vapors may cause serious

damage to the lining of the nose, throat, and lungs. Breathing difficulties may occur. Swallowing can cause severe injury leading to death. Symptoms include sore((قرحة throat, vomiting, and diarrhea.

– Ingestion of as little as 1.0 ml has resulted in perforation of the esophagus (المرئ). Contact with concentrated solution may cause serious damage to the skin. Effects may include redness, pain, skin burns.

– Eye contact with concentrated solutions may cause severe eye damage followed by loss of sight. Exposure to vapor may cause intense watering and irritation to eyes.

• Hydrochloric acid ( HCl ): – Colourless to light yellow fuming liquid when

concentrated; colourless liquid when diluted.– Short-term exposure through inhalation may cause

inflammation(التهاب) and destruction of the nasal passages, dental erosion, loss of voice, coughing, pneumonia, headaches and rapid throbbing(خفقان)of the heart.

– Small quantities of hydrochloric acid may cause irritation or burns of the skin.

– Exposure to the eyes may cause severe damage to the surface of the eye.

• Ethidium Bromide: – A fluorescent dye and a nucleic acid intercalating

agent.– EtBr is a potent mutagen (may cause genetic

damage) and can be absorbed through the skin. – Skin Contact Immediately wash the exposed area

with soap and water.

• Sodium hydroxide: – Contact with the eyes can cause serious long term

damage and its solutions are corrosive. – Contact with skin can cause irritation or severe

burns and scarring with greater exposures.– Significant heat is released when sodium

hydroxide dissolves in water.

• Ultraviolet Transilluminators:– Are designed for viewing DNA/RNA in agarose

gels stained with ethidium bromide or other dyes.

– Transparent hinged cover, with UV-blocking material to protect users from exposure to hazardous UV radiation.

The different micropipettes

)     Micro Pipette,Gilson(Useful Range Model

0.2 ∼ 2 ㎕ P20.5 ∼ 10 ㎕ P102 ∼ 20 ㎕ P20

20 ∼ 100 ㎕ P10050 ∼ 200 ㎕ P200

200 ∼ 1000 ㎕ P10001000 ∼ 5000 ㎕ P5000

1 ∼ 10 ml P10ml

• The micropipette 10µl uses white tips.

• micropipettes 20µl, 50µl, 100µl and 200µl use the yellow tips.

• Micropipette 1000µl uses

blue ones.

• Never draw any fluid into the white barrel of the pipette itself.

• Never lay a pipette down while there is fluid in the tip. The fluid may accidentally find its way into the barrel.

• Never turn the adjustment scale below or above the full range settings.

Stock solution:

concentrated solution which is

being diluted

Working solution:

diluted solution,

ready to use

Diluent: the fluid used for diluting

concentrate

Aliquot: a measured

sub-volume of original sample.

The concepts and calculations for dilutions and solutions:

Terminology and Concepts:

Dilution factor (DF): final volume / aliquot volumeConcentration factor (CF): aliquot volume / final volume

final volume = aliquot + diluent

• Example:

What is the dilution factor if you add 0.1 mL aliquot of a specimen to 9.9 mL of diluent?

• The final volume is equal the the aliquot volume plus the diluent volume: 0.1 mL + 9.9 mL = 10 mL

• The dilution factor is equal to the final volume divided by the aliquot volume: 10 mL/0.1 mL = 1:100 dilution (102)

• The Concentration Factor for this problem = aliquot volume/final volume = 0.1/(0.1 + 9.9) = 0.01 or 10-2 concentration

• This is sometimes given as:

Remember: V2 > V1 C2 < C1

C1 X V1 = C2 X V2

• Concentrated stock solutions - using "X" units: Stock solutions of stable compounds are routinely

maintained in labs as more concentrated solutions that can be diluted to working strength when used in typical applications. The usual working concentration is denoted as 1X. A solution 20 times more concentrated would be denoted as 20X and would require a 1:20 dilution to restore the typical working concentration.

• Background: The long, thick fibers of DNA store the information for the functioning of the chemistry of life.

• DNA is present in every cell of plants and animals.

• The DNA found in strawberry and onion cells for example can be extracted using common, everyday materials.

• An extraction buffer containing 1. Salt, to break up protein chains that bind

around the nucleic acids.2. Dish soap to dissolve the lipid (fat) part of

the strawberry cell wall and nuclear membrane.

3. Alcohol is used to precipitate the DNA. Because DNA is soluble in water, alcohol (ethanol) causes the DNA to precipitate and come out of the solution.

• Extraction buffer: detergent (dishwasher or shampoo)• Detergent/salt solution:

– 20 ml detergent – 20 g non-iodized salt – 180 ml distilled water

Procedure for Strawberry DNA extraction:

1. Place one strawberry in plastic bag.2. Smash/grind up the strawberry using your fist and fingers for 2 minutes. Careful not to break the bag!3. Add 10mL of extraction buffer to the bag.4. mush the strawberry in the bag again for 1 minute.5. Assemble your filtration apparatus as shown bellow.

6. Pour the strawberry slurry into the filtration apparatus and let it drip directly into your test tube.

7. Slowly pour cold ethanol(10 ml) into the tube.observe 5min

8. Dip the loop or glass rod into the tube where the strawberry extract and ethanol layers come into contact with each other. OBSERVE

CondensedDNA