Transcript Practical Of Genetics - الجامعة الإسلامية بغزة

The Islamic University
Faculty of Science
Biology Department
‫ غزة‬- ‫الجامعة اإلسالمية‬
‫كلية العلوم‬
‫قسم األحياء‬
Some of Genetic Terms
• 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.
• 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.
Some of the labs. Materials are
dangerous:
• 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(‫ (قرحة‬throat, 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(‫ )التهاب‬and destruction of the nasal
passages, dental erosion, loss of voice, coughing,
pneumonia, headaches and rapid throbbing(‫)خفقان‬of
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)
Model
Useful Range
P2
0.2 ∼ 2 ㎕
P10
0.5 ∼ 10 ㎕
P20
2 ∼ 20 ㎕
P100
20 ∼ 100 ㎕
P200
50 ∼ 200 ㎕
P1000
200 ∼ 1000 ㎕
P5000
1000 ∼ 5000 ㎕
P10ml
1 ∼ 10 ml
• The micropipette 10µl uses
• micropipettes
20µl, 50µl, 100µl
and 200µl use
the
• Micropipette
1000µl uses
• 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.
The concepts and calculations for
dilutions and solutions:
Terminology and Concepts:
Stock
solution:
Working
solution:
concentrated
solution
which is
being diluted
diluted
solution,
ready to
use
Diluent:
the fluid
used for
diluting
concentrat
e
Aliquot:
a
measured
subvolume of
original
sample.
Dilution factor (DF):
final volume / aliquot
Concentration factor (CF):
volume
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
• 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.
Plant DNA Extraction at home:
Strawberry fruit
• 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
Condensed
DNA