Ministry of Higher Education & Scientific Research

Al-Qassim Green University

College of Biotechnology
Department of Biotechnology & Genetic Engineering

ALQASSAM GREEN UNIVERSTY / Biotechnology

Principle of Biotechnology-first stage.

By .M.sc. ayser ihsan almandlawi

SAFETY RULES - Biology Laboratory

Lab. safety markers

  SAFETY RULES - Laboratory
1- You must always wear medical cloves, mask and coat.
2- Don't allow to smoke, drink and eat inside the lab.
3- Wash your hands before and after working inside the lab.
4- Keep your hands away from nose, eyes and mouth.
5- Keep your bench clean and tidy. Disinfect the bench before and
after working.
6- Don’t leave any contaminated objects around.
7- All glass wear should be sterilize by autoclave or by oven before
and after working.
8- Mouth pipetting is so dangerous, so you should use a balloon with
the pipette.
9- All the tests should be done inside the safety cabinet.
10- When you deal with antisera, you should be very careful
specially with those samples of highly infectious (HIV or HBV)
because it consider as a source of infection>
 LAB1

Laboratory Equipment's
Basic Tools in the Biotechnology Laboratory

1- Personal Protective Equipment (PPE) : refers to clothing or equipment

that is designed to protect the wearer’s body from injury or infection.
PPE includes items such as lab coats, gloves, and safety glasses

2- Erlenmeyer flask : Laboratory flasks are containers can be used for

making solutions or for holding, containing, collecting, or sometimes
volumetrically measuring chemicals, samples, solutions, etc. for chemical
reactions or other processes such as mixing, heating, cooling, dissolving,
precipitation, boiling (as in distillation), or analysis. Laboratory flask sizes
are specified by the volume they can hold, typically in metric units such as
milliliters (mL or ml) or liters (L or l). Laboratory flasks have traditionally
been made of glass, but can also be made of plastic
There are several types of laboratory flasks, all of which have different
functions within the laboratory. Flasks, because of their use, can be divided
into:

1-Multiple neck flasks, are used in more complex reactions that require the
controlled mixing of multiple reagents.

2-Distillation flasks which are intended to contain mixtures, which are subject
to distillation

3-Evaporating flasks (for rotary evaporator) centered, pear shaped, with

socket or with flange.

4-Culture flasks for growing cells are designed to improve aeration by

including baffles that aid in mixing when placed on a shaker table

1 2

3- Transfer pipet(te) are disposable plastic pipets used to transfer small

volumes of liquids. Transfer pipets are also called Pasteur pipets, teat pipets,
droppers, eye droppers and chemical droppers it designed to transfer small
amounts of liquid from one vessel to another. It has a fixed volume of 10 ml
 4- Beaker (glass wear) : Is a simple container used in many
laboratories commonly for stirring, mixing and heating liquids
commonly. Beakers are generally cylindrical in shape, with a flat
bottom. Beakers are available in a wide range of sizes, from one
milliliter up to several liters

5- Graduated cylinder : Measuring cylinder or mixing cylinder is a

common piece of laboratory equipment used to measure the
volume of a liquid. There are three size in 10mL, 50mL, 100mL

6-Volumetric pipet(te) and Graduated pipet(te) is designed to deliver a specific

volume of liquid and controller with any graduated or volumetric pipette to
accurately transfer fluids and laboratory solutions from a base . Typical
volumes are 10, 25, and 50 mL .
7- Wire / inoculating loop : instrument useful for obtaining bacterial
samples from colonies growing on (media plates -liquid media) by looped
end. It can be used and sterilized repeatedly, reducing the amount of
contaminated lab waste generated to transfer bacterial samples and other
microbiological applications.

8-Bunsen burner : is one of the most common pieces of equipment in the

laboratory that is used for heating, sterilization, and combustion Biologists
use the burner flame to sterilize tools used to handle bacteria and other
sensitive microorganisms. The gas can be natural gas (which is mainly
methane) or a liquefied petroleum gas, such as propane, butane, or a mixture
of both

https://www.youtube.com/watch?v=bRadiLXkqoU
9-Volumetric flask : (measuring flask or graduated flask )are pieces of
scientific equipment (with a calibration mark) use in laboratories. These
flasks are important for experimentation and prepartion of standard solution

10-Bench top bin: Bin holds loose lab items such as tips, micro tubes,
caps, etc.

11- liquid N2 TANK : Liquid nitrogen storage equipment is used to store biologic,
genomic ,under blew- 194c and ... Transport cryogenic industrial gases with
these LN2 Supply tanks the smaller size holding 3 liters of nitrogen up large
tanks holding 50 liters
 12- Disinfectant spray = incidin® foam are as effective as they are simple
to use. Just spray and wipe .

13-Pipette bulb = pipette filler : is used to draw liquid up into the pipette and
Pasteur pipette ,also help control the flow of liquid from the dropping bottle

14-Sterile pipet(te)=serological pipet: Sterile Serological Pipette Mainly used

for accurate measure or transfer for certain volume liquid and is widely
employed in the field of cell culture, bacteriology, clinical research, etc.

15- Stoppers= lab caps

15-Petri dish or cell-culture dish: The Petri dish is widely used in
microbiology for the cultivation of colonies of microorganisms , Scientists use
it to grow cells from animals, fungus, and diseases so they can study them.
They are usually made of glass or plastic. The glass ones can be used again if
they are heated at 160°F. Sometimes people fill them with agar, which helps
cells grow. These are called 'agar plates'.

16-Broth cultures: Broth cultures are a method of growing bacteria in a liquid

growth medium. They're used to grow and maintain cultures for a laboratory
Bacteriological culture media can be prepared as a liquid (broth), a solid
(plate media or slant media), or as a semi-solid contain( agar or gelatin)

17- Agar plate : which is a polysaccharide derived from red seaweed.

18-Sterile pipet(te)=serological pipet: Sterile Serological Pipette Mainly
used for accurate measure or transfer for certain volume liquid and is widely
employed in the field of cell culture, bacteriology, clinical research, etc.

19-Pipettor = automatic pipet: A micropipette (DIFFERENT VOLUMES) is

used to transfer small volumes of liquids in chemical, biological and medical
laboratories .Pressing on a plunger button at the top of the micropipette will
pull the liquid in, and a second press will dispense it.

•1.5 ML. EPPENDORF TUBE : 1.5ml Eppendorf tubes are sterile snap-top tubes
that are used for lots of small volume molecular biology applications
20-Pipet tip(s): use micropipette tips to dispense liquids into a well plate for
PCR assays

21-Test tube rack: They are used to hold multiple test tubes, and sometimes
pipettes, at the same time They are most commonly used when various
different solutions are needed to work in laboratory

22-Tweezers or forceps : are used for to hold or pick up small objects

23-Microscopeis: an instrument used to see objects that are too small to be
seen by the naked eye by magnify a specimen used for viewing very small
objects, such as mineral samples or animal or plant cells, typically magnified
several hundred times

24- Lab slides= microscope slides +cover slides: Microscope slides and cover
slips are used to mount, or place, specimens for study under microscope in a
way that is easier to handle and that cover slides protects them from cross-
contamination by air borne particles or others substances.
25-Staining reagents Gram: Gram staining is used for the identification and
differentiation of different types of bacteria. It differentiates Gram positive
bacteria and Gram negative bacteria on the basis of their stain retaining
abilities staining technique used in microbial and molecular biology

26- Wash bottle: A wash bottle is used to wash the slides, plates, test tubes and
round bottom flasks. Wash bottle, lab ware while experimenting laboratory

27- Funnel: used to transfer liquid in to another container or chemicals

(powders) into lab ware funnels are sometimes used to filter materials by
using filter paper
 28-Biohazard bag: used for the collection biohazard waste including gloves
specimen swabs human body fluids like semen, saliva, tissues, organs and
animal carcasses from the lab area

29- Precision scale: Used for the measurement of small samples weighing
test materials and sampling amounts, formulation, density determination,
purity analysis, quality control testing and material
 30- Absorbent tissue: removal of liquid contaminants from the skin

31- Fume hood: protects lab workers

32-Swab : Transfer of Bacterial Samples Clinical samples (biological

samples obtained from a patient) are often obtained using a sterile
swab which is then streaked onto a sterile growth medium (a plate).
 LAB 2
Preparation of
( Cell and Molecular Biology Laboratory)
 An auto clave : is a pressure chamber used to sterilize
equipment and supplies by subjecting them to high pressure
saturated steam at 121°C (249°F) for around 15–20 minutes
depending on the size of the load and the contents

 WATER BATH : It’s a container filled with heated water, used to

incubate samples in water at a constant temperature over a
long period of time, melting of substrates or incubation of cell
cultures and to enable certain chemical reactions to occur at
high temperature. Different types of water baths are used
depending on application. For all water baths, it can be used
up to 99.9°C.
  WATER DISTILLATION : Water distillation is very important in
science and research , this types are fully automatic, available
with outputs of 4 or 8 litres/hr single distilled or a double unit
producing 4 litres/hr of double distilled water

 INCUBATOR :
The applications in the incubator
1 .Growing cell cultures
2 .Reproduction of germ colonies
3. Reproduction of microorganisms such as bacteria, fungi, yeast or
viruses
4. Breeding of insects and hatching of eggs in zoology
5. Controlled sample storage 6. Growing of protein crystals
 OVEN: An oven is a thermally insulated chamber used
for the heating and drying of a substance.

 DEEP FREEZER: Used to storage of the tissue or blood samples

and DNA & RNA until application of the experimental.

 LAMINAR FLOW (HOOD) : Laminar Flow used for an individual

clean air environment is required for smaller items, commonly
used for general lab work, especially in chemical and
biological laboratories
 MICROPIPETTE (DIFFERENT VOLUMES) : A micropipette is used to
transfer small volumes of liquids in chemical, biological and
medical laboratories .Pressing on a plunger button at the top
of the micropipette will pull the liquid in, and a second press
will dispense it.

 1.5 ML. EPPENDORF TUBE : 1.5ml Eppendorf tubes are sterile

snap-top tubes that are used for lots of small volume
molecular biology applications

 DIGITAL BALANCE: The laboratory Sensitive digital balances are

used to measure an object’s mass to a very high degree of
precision, provide high readability and a high degree of
accuracy.
 MAGNETIC STIRRER HOT PLATE: Magnetic Stirrer hotplate is
generally used to heat glassware or its contents. Some hot
plates also contain magnetic stirrer, allowing the heated

 VORTEX MIXER : A vortex mixer, or vortexer , is a simple device

used commonly in laboratories to mix small vials of liquid.
Should be the test tube or other appropriate container is
pressed into the rubber cup (or touched to its edge).
REFRIGERATION CENTRIFUGE: Refrigerated laboratory centrifuges temperature
ranges as wide as -20 °C –40 °C, making them perfect for DNA, RNA, PCR or
antibody analysis. A refrigerated laboratory centrifuge can obtain rotational
speeds of over 30,000 rpm. These centrifugation systems utilize centrifuge
tips and tubes, with capacities reaching 4 x 500 ml.

 GEL ELECTROPHORESIS : The gel electrophoresis, commonly used to

separate biomolecules by their charge and frictional forces Gel
electrophoresis , a technique used by scientists

The a garose gel electrophoresis is a method of gel electrophoresis used in

biochemistry, molecular biology, and clinical chemistry to separate a mixed
population
 UV TRANSILLUMINATORS : The trans illuminators are used in molecular biology
labs to view DNA (or RNA) that has been separated by electrophoresis

 DIGITAL GEL DOCUMENTATION WITH DIGITAL CAMERAS

The trans illuminators are used in molecular biology labs to view DNA (or
RNA) that has been separated by electrophoresis and has digital camera to
viewer this bands
  NANODROP-SPECTROPHOTOMETER
Measure nucleic acid concentrations or Quantify and qualify DNA,
RNA, and protein samples with only 1-2 μL and have full-spectral data
in seconds

 CONVENTIONAL PCR INSTRUMENTS :

Thermal cycler
The thermal cycler (also known as a thermocycler, PCR machine
or DNA amplifier) is a laboratory apparatus most commonly
used to amplify segments of DNA via the polymerase chain
reaction (PCR). Thermal cyclers may also be used in laboratories
to facilitate other temperature-sensitive reactions, including
restriction enzyme digestion or rapid diagnostics. The device has
a thermal block with holes where tubes holding the reaction
mixtures can be inserted. The cycler then raises and lowers the
temperature of the block in discrete, pre-programmed steps
  THERMAL CYCLER FOR REAL TIME PCR : This method which used
in many purposes like:
1. Measure of gene expression.
2. Commonly used in studying the genomes of viruses
whose genomes are composed of RNA, such as
InfluenzavirusA and retroviruses.
3. Genetic Disease Diagnosis.
4. Cancer Detection

 THERMAL CYCLER FOR REAL TIME PCR :

 LAB3

Sterilization and Disinfection

•Sterilization : procedures are designed not just to kill replicating

microorganisms (bacteria, mycobacteria, viruses, & fungi) but also
to eliminate the more resistant spores.

There are two method of sterilization :-

1-Physical method : - by Heat -radiation - Filtration

2-Chemical method : - by gases - ethylene oxide, formaldehyde.

1 - Physical method
A-Heat method : is mostly used and reliable method of sterilization
include destruction of enzymes and other essential cell component

B- Dry Heat : is preferable , but low ( minimum) temperature of 160

C maintained for 1 h is required dry heat bacteria endotoxin
which are difficult to eliminate. ex : Red heat – Flaming – HOT AIR
OVEN
C-Moist Heat ( for fluids and perishable items) by use of steam in
the range at 121 c for 15 -20 min under pressure .

ex: Dry steam – Auto clave –Boiling water .

Auto clave : is the process of sterilizing subsistence and

equipment with the use of high temperatures pressure up to
(121°C – 134 C) for about 15 minutes depends on the material .
The high temperatures pressure destruction of all microorganisms
including bacteria that are resistant, bacterial spores, protozoans, p,
viruses and fungi that are present in ( fluids – glass wear – media).

1- Auto clave

2- Pressure cooker

B- Radiations: includes (gamma irradiation, electron irradiation

and x-ray irradiation and UV light).

 Use: These radiations target (Microbes DNA) and destroyed it

 Destroy all microorganisms during sterilization and useful
method for industrial sterilization
C- Filtration : The process in which solid particles in a liquid or fluid
are removed by the use of a filter medium without using heat .

Ex - Filter flask

2-Chemical method: mostly used to sterilization and disinfection

Alcohols- Ethylene Oxide – Ozone- Formaldehyde - Hydrogen
Peroxide.
 Disinfection: Disinfection describes a process that
eliminates many or all pathogenic microorganisms, except
bacterial spores -Aseptic techniques are used to prevent
contamination of surgical instruments, medical personnel, and the
patient during surgery. Aseptic techniques are also used to prevent
bacterial contamination in food industry

 Disinfection methods
A- Alcohols and Phenols , Aldehydes , Halogens and hydrogen
peroxide 6%.
B-Physical methods:
-Boiling & Pasteurization
-UV(ultraviolet radiation)

Q- why is 70% alcohol better than 100%?

-Pure alcohol 100% coagulates protein in cell wall that it is contact.
The alcohol 100% will go through the cell wall of the organism in all
direction, coagulating the protein just inside the cell wall. The ring of
the coagulated protein would then stop the alcohol from penetrating
farther from the cell, and no more coagulation would take place. At
this time the cell would become inactive but not dead. Under the
favorable conditions the cell would then begin to function. If 70
percent of alcohol is poured to a single celled organism, the diluted
alcohol also coagulates the protein, but at a slower rate, so that it
penetrates all the way through the cell before coagulation can block
it. Then the entire cell is coagulated and the organism dies.
 Lab 4
DNA EXTRACTION

 DNA EXTRACTION : is a routine procedure used to isolate

DNA from the nucleus of cells. Scientists can buy ready‐
to‐use DNA extraction kits.

 These kits help extract DNA from particular cell types or

sample types. However, they can be expensive to use
routinely, so many labs have their own methods for DNA
extraction

 DNA EXTRACTION Many different methods and

technologies are available for the isolation of genomic
DNA.
 All methods involve: Disruption and lyses of the
starting material followed by removal of proteins
and other contaminants and finally recovery of the
DNA
  METHODS OF DNA EXTRACTION:
1-Ethanol precipitation

 Usually by ice-coldethanol or isopropanol.

Since DNA is insoluble in these alcohols, it will
aggregate together, giving apellet up on
centrifugation. Precipitation of DNA is improved
by increasing of ionic strength, usually by
addingsodium acetate.

2-Phenol–chloroform extraction
The phenol which denatures a proteins in the sample. After
centrifugation of the sample, denaturated proteins stay in
the organic phase while aqueous phase containing nucleic
acidis mixed with the chloroform that removes phenol
residues from solution.
3-Minicolumn purification
That relies on the fact that the nucleic acids may bind
(adsorption) to the solid phase (silica or other)
depending on thepHand the salt concentration of the
buffer.

 DNA EXTRACTION depends on many factors:

A. The quantity and molecular weight of the DNA
B. The purity reqiredfor application
C. The time and expense

 SAMPLE COLLECTION :
A- Source: Sample can be isolated from any
living or dead
organism
Common sources for DNA isolation include:
A- Source: Sample can be isolated from any
living or dead organism
Common sources for DNA isolation include:
-Whole blood -Buffy coat
- Bone material - Buccal cells
- Cultured cells - Amniocytes or amniotic fluid
- Sputum, urine, CSF, or other body fluids
B. Sample age:
May be fresh or has been stored .Stored sample can come from:
- Archived tissue samples , - Frozen blood or tissue (biopsy
material) , - Exhumed bones or tissues &
- Ancient human sample. - Dried blood spots
Steps
- Lyses of the cells - Removal of contaminants Includes
- Proteins
- RNA
- Other macromolecules
- Concentration of purified DNA

STEP 1. BREAKING CELLS OPEN TO RELEASE THE DNA

A. Put into a solution containing salt. The positively
charged sodium
ions in the salt help protect the negatively charged
phosphate
groups that run along the backbone of the DNA.
B. A detergent is then added to breaks down the lipids
in the cell
membrane and nuclei. DNA is released as these
membranes are
disrupted.
The cells in a sample are separated from each other,
often by a physical means such as grinding or vortexing
STEP 2. SEPARATING DNA FROM PROTEINS AND OTHER
CELLULAR DEBRIS To get a clean sample of DNA, it’s necessary to
remove as much of the cellular debris as possible, by a variety of
methods.
A. Protease ( protein enzyme) is added to degrade DNA associated
proteins and other cellular proteins.
B. Some of the cellular debris can be removed by filtering the sample
STEP 3. PRECIPITATING THE DNA WITH AN ALCOHOL
Finally, ice‐cold alcohol (either ethanol or isopropanol) is carefully
added to theDNA sample. The DNA is soluble in water but
insoluble in the presence of salt and alcohol. By gently stirring the
alcohol layer with a sterile pipette, aprecipitate becomes visible and
can be spooled out. If there is lots of DNA, you may see a stringy,
white precipitate.

STEP 4. CLEANING THE DNA

The DNA sample can now be further purified (cleaned). It is then
resuspended in a slightly alkaline buffer to cleaning up DNA from
aqueous solutions to remove buffer salts, enzymes or other
substances that could affect on other applications.

STEP 5. CONFIRMING THE PRESENCE AND QUALITY OF THE

DNA For further lab work, it is important to know the concentration
and quality of the DNA.
 Optical density readings taken by aspectrophotometer can be used
to determine the concentration and purity of DNA in a sample.
Alternatively, gel electrophoresis can be used to show the presence
of DNA in your sample and give an indication of its quality.