MOLECULAR BIOLOGY AND DIAGNOSTICS

LABORATORY

JUSTIN KIM C. VERGARA, RMT, MPH

COLLEGE OF MEDICAL LABORATORY SCIENCE
OUR LADY OF FATIMA UNIVERSITY- VALENZUELA
 The Molecular Laboratory Set-Up

• Needs adequate space, which

ideally consists of three
separate areas:

– Reagent preparation room

– Sample preparation room
– PCR room

OUR LADY OF FATIMA UNIVERSITY- COLLEGE OF MEDICAL LABORATORY SCIENCE

The Molecular Laboratory Set-Up

OUR LADY OF FATIMA UNIVERSITY- COLLEGE OF MEDICAL LABORATORY SCIENCE

Why separate areas?
• Introduction of unwanted nucleic acids into specimen
• Repeated amplification of the same target sequence leads to
accumulation of amplification products in the laboratory
environment
– A typical PCR generates as many as 1B copy of target sequence
– Aerosols from pipettes will contain as many as 1 M amplification
products
– Build-up of aerosolized amplification products will contaminate the
laboratory
CONTAMINATION IN A PCR LAB

• Amplification product contamination

• Cross contamination between specimens
• Laboratory surfaces
• Ventilation ducts
• Reagents/supplies
• Hair, skin, saliva, and clothes of lab personnel
CONTAMINATION CONTROL PROGRAM

What happens if  Incorrect results

there is lack  Require extensive clean-up
contamination  Loss of credibility
control?  Impact on economy and performance
Biosafety Cabinet

• Method of protection: HEPA

Filters
• Protection to the user,
product, and environment
• Decontamination: 10%
Bleach→70% Ethanol
Biosafety Cabinet Class I
• Air goes inside cabinet without
filtration, comes out of
cabinet with filtration
• No product protection needed
– Opening suspicious mail
– Running Centrifuges
– Housing fermenters
– Aerating cultures
Biosafety Cabinet Class II
• Provides personnel, product, and environmental
protection
• Does not provide absolute containment, can only
provide if these 3 aspects are maintained:
– Open front with inward airflow
– Downward HEPA Filtered Laminar Airflow
– HEPA Filtered Exhaust Air
Biosafety Cabinet Class II
• Type A1
– Plenum is under positive pressure
– Without CANOPY
• Type A2
– Plenum is under negative pressure
– Two types:
a. Without Canopy
b.With Canopy (can minimize
release of chemical fumes in the
room)
Biosafety Cabinet Class II
Biosafety Cabinet Class II
• Type B1
– 40% Air recirculated
– 60% Exhausted outside thru
hard ducting (HEPA Filters)
• Type B2
– 100% Air exhausted via hard
ducting (HEPA Filters)
Biosafety Cabinet Class III
• Gas tight, under negative pressure
• NO recirculation of air
• HEPA Filtered air supply
• Exhaust air: Double HEPA-filtered or HEPA-Filtered
and Incinerated
• Operation inside thru Rubber Gloves
• Cabinet connected to a double-door autoclave and/or
chemical dunk tanks to sterilize/disinfect all exiting
materials
Dry Bath
• For heating of samples
•Physical Inactivation
Two kinds:
– Digital: Uses
Microchip
technology, controlling the
temperature using digital
interface
–Analog: Does not have a
Microchip technology
Electrophoresis apparatus
• DNA, RNA, Protein separation
– Size
– Electrical Charge
• Used for:
– Detection
– Purification
Microcentrifuge/Microfuge

• Principle: Sedimentation by
Centrifugal force
• For samples less than 2 mL
• Speed: Up to 17000 x g
• Max samples: 24 per run
Micropipettes and
Micropipette Tips
Principle: Plunger depressed and when released, liquid
is drawn into disposable plastic tip.
For volume measurements <1000 uL
Sizes:
– P2(0.2–2uL)
– P10 (1-10 uL)
– P20 (2-20 uL)
– P100 (20-100 uL)
– P200 (20-200 uL)
– P1000 (100-1000 uL)
Micropipette Tips
PCR Cabinet
• For preparation of PCR Master Mixes
• Product protected from contamination
• Air flow: filtered first before going
inside the cabinet
• Delivers HEPA-Filtered Air across work
surface
– Facilitates worker exposure to
materials in use
PCR Machine
• Principle: DNA Replication
• Technically an IN VITRO method of
DNA Replication
– If sample is RNA: Reverse
Transcriptase
• One run = ~40 cycles
– Run time: 1 hour 30 mins
–2
hours
• Three major cycles:
– Denaturation
– Annealing
– Extension
PCR Machine
Refrigerato
r
• Temperature: 2 to 6 C
• For reagents and samples
that will be processed
immediately
• Storage only until 24
hours or less
Ultra low Freezer
• Temperature: -60 to -80 C
• For fast freezing of samples
• What to avoid: Freeze-thaw
cycles
• Best for:
– Eluates
– Storage of specimens
Spectrophotometer

• Principle: Measures
light absorbance across
the visible and
ultraviolet ranges of the
spectrum.
• Used for: Quantification
of analytes
Vortex Mixer
• For mixing of small
bottles of liquid
• For suspension or re-
suspension of cells
• Principle: transmission
of vortex motion
 Molecular Laboratory Equipment

Biosafety Cabinet Autoclave Refrigerator: 4oC -20oC Freezer

freezer: -18oC

OUR LADY OF FATIMA UNIVERSITY- COLLEGE OF MEDICAL LABORATORY SCIENCE

 Molecular Laboratory Equipment

Analytical Balance Vortex pH Meter Spectrophotometer

OUR LADY OF FATIMA UNIVERSITY- COLLEGE OF MEDICAL LABORATORY SCIENCE

 Molecular Laboratory Equipment

Thermocycler Dry bath Electrophoresis Microcentrifuge

Apparatus

OUR LADY OF FATIMA UNIVERSITY- COLLEGE OF MEDICAL LABORATORY SCIENCE

 Molecular Laboratory Equipment

UV Transilluminator Gel Doc Microwave oven Refrigerate

d
Centrifuge

OUR LADY OF FATIMA UNIVERSITY- COLLEGE OF MEDICAL LABORATORY SCIENCE

 Molecular Laboratory Equipment
Pipette type Volumes (μL) Tip color

P10 0.5–10 white

P20 2–20 yellow

P200 20–200 yellow

P1000 200–1000 blue

P5000 1000–5000 white

Pipettors Pipette Tips

OUR LADY OF FATIMA UNIVERSITY- COLLEGE OF MEDICAL LABORATORY SCIENCE

 Molecular Laboratory Equipment
Microcentrifuge tubes

0.2-ml for PCR

0.5-ml

1.5-ml

Tip Racks Microcentrifuge

Tubes
Eppendorf

OUR LADY OF FATIMA UNIVERSITY- COLLEGE OF MEDICAL LABORATORY SCIENCE

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