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LAB OF MOLCULAR BIOLOGY MANUAL

• THE DESIGN LABORATORY
• FEATURES OF LABORATORY
• LIST OF INSTRUMENTS AND EQIPMENT
• THE EXPIERMENTS IN MOLCULAR BIOLOGY LABORATORY
• GENERAL SAFETY RULES FOR QUALITY CONTROL LABORATORY
• CAMPUS BIOMEDICAL WASTE MANAGEMENT STANDARD OPERATING
PROCEDURE (SOP)
• REFERENCE

REPORT OF MOLCULAR BIOLOGY LABORATORY

QUALITY AND CONTROL LABORATORY – DR . MODHI ALABALAWI
WRTTIEN BY MASTER STUDENT – MANAL FAHAD ALBALAWI – 431008333
109

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SECTIONS section A - study - research - storage

OF
LABORATORY section B - expirement region

section C - expirement region

WORK Flexible Workflow Solution For Best Lab Routine For

FLOW
Every Laboratorian

PROCESS OF A - Purification
WORK FLOW
IN B - Quantification
LABORATORY
C - Preparation

D - Amplification

E - Storage

FEATURES Genotyping For Molecular Epidemiology Studies

OF
Molecular Diagnostics For Infectious / NCDs
LABORATORY
WGS Using NGS For Small Genome ( MTB , MRSA , HIV virus , etc )

DNA/RNA Extraction and QC

Amplicon Sequencing For 16 S And 18S_ITS S For Bacterial

Eukaryotic Pathogen Profiling For Metagenomics

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Notic : The distribution of equipment in the laboratory depends on the work flow, which is
determined by the laboratory workers, So I wrote it down below with a video of the mechanism
of work and use without distributing it to laboratory benches in the design

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Section A à laboratory disk + Files cabinet + Closet

Section B à Work space 1 + Shelves

Section C à Shelves + Work space 2 + Work space 3

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INSTRUMENTS A. PPE
AND
EQIPMENTS B. Volume Measuring Equipment

C. Analyzers

D. Others

A PPE - Personal Protective Equipment

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B Volume Measuring Equipment

B l - Non-Volumetric For à mixing and storing – not calibrated

E.g. break and Erlenmeyer flasks

B ll - Exact Volumes - calibrated

• volumetric flasks – specific concentrations
• graduated cylinders – liquid above 50 ml
• Serological pipettes-0.1-50ml
• Syringes – transferring liquid chemicals
• Micropipettes – 0.2UL – 5mL
• Glass Hamilton syringes – alternative for plastic UL pipette

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B lll - Micropipettes

B lV - Microscopes

Fluorescence microscope

à target proteins, cells

and cellular components

green fluorescents

protein (GFP)

Electron microscope à

higher resolution – require vacuum – cannot be used on live samples

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C Analyzers

1. Microplate Reader https://youtu.be/0OSHJi-5UnQ

2. PCR apparatus ( Polymerase Chain Reaction Machine )

https://youtu.be/-R_-bnbOGMI

3. Electrophoresis Apparatus https://youtu.be/uKwSxPgqS3U

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4. Gel Documentation System https://youtu.be/ZB2SGwn4ia0

https://youtu.be/UAksP-E4puM

5. Spectrophotometer https://youtu.be/xHQM4BbR040

6. Chromatography HPCL https://youtu.be/51DSl8BHUjg

https://youtu.be/YaabRXcm4M4

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D others ________________________________________________________________

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1. Mixers And Sonicators https://youtu.be/hLNk-1cdJb4

2. Ph Meter https://youtu.be/vwY-xWMam7o

3. Centrifuge https://youtu.be/NqVaMiTI8Uw

4. Microwave Oven

5. Autoclave https://youtu.be/0dlDf8HiiQw

6. Hot Plate With Magnetic Stirrer https://youtu.be/RRdL_xMAdaQ

7. Water Baths https://youtu.be/7Hi4_xHMVjw

8. Biological Safety Cabinets https://youtu.be/96-aZLom340

9. Fume Hoods https://youtu.be/WlEgeQoqZkQ

4. THE EXPIERMENT
MOLECULAR BIOLOGY

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• DNA Extraction
https://youtu.be/tcPgdR9_t64
https://youtu.be/1PisbDHKXTU
• RNA Extraction
https://youtu.be/9WSkt9JEok8
• cDNA Synthesis
https://youtu.be/gXE-9T9Mj4c
• Conventional PCR
https://youtu.be/QYpX94prb0A
https://youtu.be/MxDgPFNjkbw
• Real-Time PCR
https://youtu.be/Dho2ddDmam8
• 2D Protein Electrophoresis (Isoelectric Point Focusing, PAGE)
https://youtu.be/Z2U0_BsVXnU https://youtu.be/ZQgR9Ww9xi4
https://youtu.be/S_h76Vl0T6c https://youtu.be/mkMPx49QZtw
https://youtu.be/VjrhnMRBxcI https://youtu.be/SaU1qX37nPM
https://youtu.be/8p-zBJfJZ_0 https://youtu.be/4WuUZJc5fOA
https://youtu.be/Wm6svEu-H9A https://youtu.be/Sm_HTFWWXGw
https://youtu.be/atnTfPVCxH8 https://youtu.be/sz3BN7C_FSI
https://youtu.be/G3auxOfvQbg
• Protein Electrophoresis (Polyacrylamide Gel Electrophoresis – PAGE)
https://youtu.be/pnBZeL8nFEo
• Western Blot
https://youtu.be/yUstng0npaY
• DNA Sequencing
https://youtu.be/bJfed2B2Pzk https://youtu.be/rA8MUR4pqNE
• DNA Microarray
https://youtu.be/0ATUjAxNf6U

IMMUNOLOGY
• ELISA – Sandwich
https://youtu.be/CaEwcf5ntWw

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• ELISPOT Assay (Dot ELISA)

https://youtu.be/05f8A1ucfSI
• Immunofluorescence Assay
https://youtu.be/QdjIfOl2zwQ
• Flow Cytometry – Cell Cycle
https://youtu.be/RbisI3fws_g

TOXICOLOGY
• MTT Viability Assay
https://youtu.be/vn6enA6lSKs
• In Vitro Cell Viability by the Lactate Dehydrogenase Assay (LDH)
https://youtu.be/Q5FmzQrKCbY
• In Vitro Cell Viability by the Alamar Blue Assay
https://youtu.be/Jz2Asfls5Hs
• In Vitro Histone H2AX Phosphorylation Assay
• In Vitro 80HdG DNA Adduct Assay
• In Vitro Bromodeoxyuridine (BrdU) Assay
• In Vitro Mammalian Cells COMET Assay Single Cell Gel
Electrophoresis (SCGE) Assay
https://youtu.be/EWrkB16Zf5I https://youtu.be/drbMxbFf3TM
https://youtu.be/kAPE6ATqkyI https://youtu.be/XluBt79byzo
https://youtu.be/Se56S2HwMaE https://youtu.be/g7-wEylfGvY
https://youtu.be/XeXUaowjZPg https://youtu.be/vCT0ob_1ZK4
https://youtu.be/oghp3--21x4

BIOCHEMISTRY
• Bradford Assay
https://youtu.be/EhinSM9FZCg
• High Performance Liquid Chromatography – Protein (HPLC)
https://youtu.be/kz_egMtdnL4
• Polarographic Oxygen Respirometry (Bioenergetics)
https://youtu.be/xtZhgu2EDGA

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CELL CULTURE
• Production of Monoclonal Antibodies (mAB) – Hybridomas
Technique

5. GENERAL SAFETY RULES FOR QUALITY CONTROL LABORATORY

General rules for safe working in accordance with national regulations and SOPs normally include
the following requirements:

• safety data sheets should be available to staff before testing is carried out;
• smoking, eating and drinking in the laboratory should be prohibited;
• staff should be familiar with the use of fire-fighting equipment, including fire
extinguishers, fire blankets and gas masks;
• staff should wear laboratory coats or other protective clothing, including eye protection;
• special care should be taken, as appropriate, in handling, for example, highly potent,
infectious or volatile substances;
• highly toxic and/or genotoxic samples should be handled in a specially designed facility to
avoid the risk of contamination;
• all containers of chemicals should be fully labelled and include prominent warnings (e.g.
“poison”, “flammable”, “radioactive”) whenever appropriate;
• adequate insulation and spark-proofing should be provided for electrical wiring and
equipment, including refrigerators;
• rules on safe handling of cylinders of compressed gases should be observed and staff
should be familiar with the relevant colour identification codes;
• staff should be aware of the need to avoid working alone in the laboratory; and
• first-aid materials should be provided and staff instructed in first-aid techniques,
emergency care and the use of antidotes.
• Protective clothing should be available, including eye protection, masks and gloves. Safety
showers should be installed.
• Rubber suction bulbs should be used on manual pipettes and siphons.
• Staff should be instructed in the safe handling of glassware, corrosive reagents and
solvents and particularly in the use of safety containers or baskets to avoid spillage from
containers.
• Warnings, precautions and instructions should be given for work with violent,
uncontrollable or dangerous reactions when handling specific reagents (e.g. mixing water
and acids, or acetone–chloroform and ammonia), flammable products, oxidising or
radioactive agents and especially biological such as infectious agents.
• Peroxide-free solvents should be used.
• Staff should be aware of methods for the safe disposal of unwanted corrosive or products
by neutralisation or deactivation and of the need for safe and complete disposal of
mercury and its salts.

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• Poisonous or hazardous products should be singled out and labelled appropriately, but it
should not be taken for granted that all other chemicals and biologicals are
safe. Unnecessary contact with reagents, especially solvents and their vapours, should be
avoided.
• The use of known carcinogens and mutagens as reagents should be limited or totally
excluded if required by national regulations.
• Replacement of toxic solvents and reagents by less toxic materials or reduction of their
use should always be the aim, particularly when new techniques are developed.

6. CAMPUS BIOMEDICAL WASTE MANAGEMENT STANDARD

OPERATING PROCEDURE (SOP)
NOTE: This SOP for biological waste management does not supersede the requirements for
radioactive and/or hazardous chemical waste management. If your waste biomedical waste is
mixed waste (i.e. contaminated with chemicals or radioactive materials) that waste will need to
be disposed of according to the recommendations of those safety offices.

Types of Biomedical Wastes

• Solid Biomedical Waste (i.e. plasticware, tubing, pipette tips, gloves)

• Sharps Waste (i.e. needles, glass, scalpels, razor blades)
• Liquid Biomedical Waste (i.e. cultures, stocks, vaccines)
• Pathological Waste (i.e. human tissues, blood and other body fluids)
• Animal Waste (i.e. carcasses, tissues, bedding)
• Chemotherapy Agent Wastes (i.e. any disposable material that has come into contact
with cytotoxic/antineoplastic agents including gloves, vials, IV tubing)
• Discarded Contaminated Equipment (i.e. centrifuges, shakers, biosafety cabinets)

Solid Biomedical Waste

Solid biomedical waste should be placed in a biohazard, red-bag lined box (shown to the right),
provided by Environmental Services. To request additional biohazardous waste containers, or to
request waste pick-ups,

• Liquids or soggy materials should NOT be placed inside the Stericycle red-bag lined boxes.

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• No loose sharp items should be placed in these containers which would puncture the red
bags.
• Do not overfill the boxes. The boxes should be removed when 2/3 full. These containers
have a 50 lb. maximum weight limit.
• Any material of higher hazard (≥BSL2) should be decontaminated prior to placement into
these containers. BSL2+ designated laboratories should follow this practice.
• Biohazard waste boxes or containers should not be left in unsecured areas (e.g., in
hallways or on loading docks) where non-trained personnel or personnel with unknown
health status may encounter them. Biohazardous waste, by definition, is hazardous
material and must remain secured at all times
• Items which are not biological waste or potentially contaminated with biological
materials should NOT be placed in the biohazardous waste containers.
• Do not combine the contents of one box into another or lift the bag out of the box.
• Alternate intermediary biohazard waste containers can be used, provided that: ¬
Containers are clearly demarked as biohazardous waste with color-coded labels (e.g., by
using red bags or biohazard stickers).
• The waste in these containers are removed promptly after use and not allowed to linger
in these intermediary waste containers.
• Containers are capable of being decontaminated and are decontaminated often.
• No sharps are disposed in these intermediary containers (sharps must be directly placed
into authorized sharps containers).
• The waste is transferred to the authorized waste receptacle by the laboratory staff (not
the Environmental Services staff).

Sharps Waste
• There are two types of sharps containers available upon request from Environmental
Services. To request additional sharps waste containers.
• Each of these containers all comply with OSHA BBP standard requirements dictated for
sharps containers. These are: closable, puncture-resistant, leak-proof on the sides and
bottom, labeled and/or color-coded.
• Soggy items and small amounts of liquids (e.g., a few ml of blood remaining in a tube) can
be disposed in these containers. Larger volumes of liquids should be handled as liquid
waste.
• Containers should be easily accessible and located close to work areas where sharp
materials are used.
• The containers must be maintained upright.

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• These containers need to be replaced routinely. Do not allow these containers to become
overfull. These containers should be closed and removed when 2/3 full.
• Do not remove the lids from these containers or force objects into them.

Liquid Biomedical Waste

• MUST be decontaminated before disposal according to your laboratory or clinic SOPs.

• Liquids that have been autoclaved or decontaminated using bleach can be disposed of
using the sanitary sewer system.
• Special procedures may be required to deactivate toxins, prior to disposal.
NOTE: Decontamination with a chemical other than bleach may require special disposal
procedures; contact the Chemical Safety Office for guidance.
• Caution should be taken not to dispose of any material which may clog sewer disposal
pipes.
• All vacuum aspirator traps/flask MUST have either a filter or secondary overflow two flask
disposal system to protect vacuum lines from entry of biological fluids.

Pathological Waste

• Any item which is identifiable as a human or animal body part or an animal carcass needs
to be disposed pathological waste. These items must be incinerated.
• All animal carcasses be packaged and labeled according to LAS/IACUC compliant
procedures and placed in one of the necropsy freezers (e.g., in CB- 1344, CA-1105, CA-
1126, CA-1135, BG-1135A, CL-1119 or CL-1133) or in the cold storage room (CB-3100).

Animal Waste

• Proper disposal of animal carcasses is described in the pathological waste section.

• Animal bedding is disposed of based on the hazards. Contact LAS for the proper disposal
procedures. Animal bedding generated from ABSL1 or conventional animal housing can
be disposed of in the regular trash. Bedding generated from animals housed in an ABSL2
facility will need to be collected and autoclaved prior to exiting the ABSL2 facility.

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NOTE: Animal bedding that may contain chemotherapeutic agents, other high hazards
chemicals or radioactive materials may require special disposal procedures (i.e. bedding
contaminated with chemotherapeutic agents cannot be autoclaved).

Chemotherapy Waste

• Chemotherapy wastes must be incinerated, as per Georgia EPD Chapter 391-3- 4-15.
While these agents are often used in clinical therapy, many of these agents may also be
used for in vitro and in vivo animal research applications (e.g., Actinomycin D, Mitomycin-
C, Streptozotocin, Bleomycin, GM-CSF, Interleukin-2, INF-α, Gleevec).
• According to GA EPD, chemotherapy wastes include:
“Any disposable material which has come in contact with cytotoxic/antineoplastic agents
(agents toxic to cells) and/or antineoplastic agents (agents that inhibit or prevent the
growth and spread of tumors or malignant cells) during the preparation, handling, and
administration of such agents. Such waste includes, but is not limited to, masks, gloves,
gowns, empty IV tubing bags and vials, and other contaminated materials. The above
waste must first be classified as empty which means such quantity that it is not subject to
other federal or state waste management regulations prior to being handled as
biomedical waste.”
NOTE: "Empty" is generally defined as containing less than 3% by weight of the total
capacity of the container.
• Stock solutions of these chemicals and items that are heavily contaminated must be
disposed of as hazardous waste.

Discarded Contaminated Equipment

• Equipment that has come into contact with biohazardous materials will need to be
decontaminated prior to disposal, transport, surplus, etc. The method of
decontamination is dependent upon the equipment and the biohazard that the
equipment has come into contact with.
• Your laboratory SOPs should indicate a disinfection method suitable for the agents used
in your laboratory.
• Be sure to follow the instructions for the disinfectant (i.e. concentration, contact time).
• The need for gaseous decontamination of a Biosafety cabinet (BSC) must be evaluated by
• the Biosafety Office prior to moving. Should a BSC require decontamination,
arrangements must be made in advance with Laboratory Equipment Services (LES).

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7. REFERENCE
- EMBL – the European molecular biology laboratory
- Praxis Labs
- amrita university
- royal society of chemistry
- eroglu lab
- euryopia union integrated project
- ANAMOL LABORATORIES PRIVATE LIMITED
- ABNOVA
- dr german rosas -acosta Texas university
- COSTAATT LABS , india
- Nepal university
- A. Malcolm Campbell, Ph.D , 2017 NC Community Colleges and BioNetwork
- the Jackson laboratory
- cell singnaling technology
- Dr. Lexa Scupham , virology laboratory for center veterinary biologics , USDA
- Food and Agriculture Organization of the United Nations FAO
- Bio-Rad Laboratories
- NC State Undergraduate Organic Chemistry Teaching Laboratories - S.M.A.R.T. Lab Videos
- Bio Network NC community collages
- National Institutes of Health (NIH)
- WHO good practices for pharmaceutical Quality Control laboratories.
- AUGUSTA university, Goss Lane, Augusta

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