GENERAL LABORATORY PROCEDURES, EQUIPMENT USE AND SAFETY CONSIDERATIONS Flashcards
involves all the measures taken by the laboratory worker, laboratory owner, institution and regulatory agencies to eliminate potential harm to human health and well-being.
Laboratory safety
Importance of Laboratory safety
to eliminate potential harm to human health and well-being.
is a matter of personal responsibility.
safety
It is the responsibility of each person in the lab to…: (4)
to know and follow basic laboratory safety rules
to understand how to safely operate equipment
understand the hazards of materials they are working with and
to work to reduce potential risks
SAFETY PROCEDURES (4)
Chemical
Ultraviolet light
Electricity
General housekeeping
MSDS
Material safety data sheet
Describe
Material Safety Data Sheets or
MSDS-
a document that contains
information on the potential
hazards and how to work safely
with the chemical product.
- carcinogen
- potential neurotoxin
- can cause severe burns
Ethidium bromide
Acrylamide
Phenol
Ethidium bromide -
Acrylamide -
Phenol -
carcinogen
potential neurotoxin
can cause severe burns
Material Safety Data Sheets or MSDS.
This information contains…
the chemical name
Chemical Abstracts Service (CAS) number and health hazard
data including first aid treatment
physical data
fire and explosion hazard data
reactivity data
spill or leak procedures
and any special precautions needed when handling the chemical
What to do when exposed to UV light?
always wear appropriate eye protection when using UV lamps
UV light can cause…?
Eye irritation
The voltages used for_electrophoresis are sufficient to cause_______.
What to do?
electrocution
Always turn off the power supply and unplug the leads before removing a gel
GENERAL HOUSEKEEPING
- Keep your own area clean
- All solutions and everything
stored in an incubator, refrigerator, etc. must be labelled. - Unlabeled material found in the
refrigerators, incubators, or
freezers may be destroyed.
Preparation of Solutions
A. Calculation of Molar, % and “X” Solutions
B. Preparation of Working Solutions from Concentrated Stock Solutions
C. Steps in Solution Preparation
D. Glassware and Plastic Ware
Dirty test tubes,
bacterial contamination and
traces of detergent can….
inhibit reactions or degrade nucleic acid.
Glassware should be rinsed with_____ and autoclaved or baked at _____ for ____
distilled water
150 C for 1 hour
For experiments with____, glassware and solutions are treated with diethyl-pyrocarbonate to inhibit_____ which
is resistant to autoclaving.
RNA
RNases
For experiments with RNA, glassware and solutions are treated with_______ to inhibit RNases which is resistant to_____.
diethyl-pyrocarbonate
autoclaving
•______ are turbid and are resistant to many chemicals, like phenol and chloroform
•_________ are clear and not resistant to many chemicals.
Polypropylene tubes
Polycarbonate or Polystyrene tubes
• Polypropylene tubes are….
• Polycarbonate or Polystyrene tubes are…
turbid and are resistant to many chemicals, like phenol and chloroform
clear and not resistant to many chemicals.
Disposal of Buffers and Chemicals
- Any contaminated, solidified
agar or agarose should be
discarded in the_____, not in the
___, and the bottles rinsed well.
trash; sink
Disposal of Buffers and Chemicals
- Any media that becomes
contaminated should be
promptly______ before
discarding it.
autoclaved
Disposal of Buffers and Chemicals
3._______ reagents, should be
used in a fume hood and all
organic waste should be
disposed of in a_____, not in _____
Organic
labelled container, not in the
trash or the sink
Disposal of Buffers and Chemicals
4.________ is a mutagenic
substance that should be heat
treated before disposal and
should be handled only with
gloves. It should be disposed of
in a______.
Ethidium bromide
labelled container
Disposal of Buffers and Chemicals
- Dirty glassware should be rinsed.
Bottle caps, stir bars and spatulas
should not be placed in the bins
but should be washed with….
hot soapy water,
rinsed well with hot
water, and
rinsed three times with distilled water
Disposal of Buffers and Chemicals:
- Discard contaminated, solidified agar/agarose in the trash, not the sink, and rinse bottles well.
- Autoclave contaminated media before disposal.
- Use organic reagents in a fume hood; dispose of organic waste in labeled containers, not in the trash or sink.
- Handle ethidium bromide with gloves, heat-treat before disposal, and discard in a labeled container.
- Rinse dirty glassware; wash caps, stir bars, and spatulas with hot soapy water, rinse with hot water, and rinse three times with distilled water.
EQUIPMENT (MMVP TAG)
Micropipette
Micro centrifuge
Vortex
pH meter
Thermal cycler
Autoclave
Gel electrophoresis apparatus
Most of the experiments
conducted in the laboratory will
depend on your ability to
accurately measure volumes of
solutions using______.
micropipettes
is an instrument that measures the potential difference between a reference electrode and a glass electrode, often combined into one combination electrode.
pH meter
This instrument amplifies target
nucleic acid sequences into millions
of copies using the polymerase
chain reaction (PCR) technique
Thermal cycler
This equipment facilitates the
separation of biomacromolecules,
such as DNA, RNA, or proteins,
based on their molecular size
Gel electrophoresis
A device that mixes liquids in
small containers by creating a
_____, or whirlpool, in the
liquid.
vortex
A compact laboratory device
that spins liquid samples at
high speeds to separate their
components
Micro-centrifuge
physical method for disinfection and sterilization.
They work with a combination of steam, pressure and time. _____ operate at high temperature and pressure in order to kill microorganisms and spores
Autoclaves
- Heavy Metals Promote______
• _______are the chemical links between the sugar and phosphate groups in DNA’s backbone.
• ______(like lead, mercury, or copper) can react with DNA and cause these bonds to break, damaging the DNA structure.
• Why is this important? If these bonds are broken, the DNA strands become fragmented, making the DNA unusable for experiments like PCR or sequencing.
Phosphodiester Breakage
Phosphodiester bonds
Heavy metals
- Free Radicals and Phosphodiester Breakage
• Free radicals are highly reactive molecules formed by chemical reactions or radiation.
• These radicals can attack DNA, breaking the phosphodiester bonds and damaging the backbone.
• Example: Radiation exposure (like X-rays) can generate free radicals in cells.
• Why is this important? Free radical damage can lead to_____, which is a problem in research and contributes to diseases like cancer.
DNA degradation
- UV Light at_____ Causes DNA Damage
• UV light, especially at _____, is absorbed by DNA and causes damage.
• This damage includes the formation of lesions like______, where two thymine bases bond together incorrectly.
• This disrupts the DNA’s structure, causing loss of biological activity (e.g., the DNA can’t replicate or transcribe properly).
• Why is this important? If DNA is exposed to UV light during an experiment, its quality can be ruined.
260 nm
thymine dimers
- Ethidium Bromide and Photooxidation of DNA
• __________is a dye commonly used to visualize DNA under UV light, but it’s also a mutagen.
• When EtBr interacts with DNA in the presence of visible light and oxygen, it causes photooxidation (a chemical reaction that damages DNA).
• This reaction produces oxidation products that lead to phosphodiester bond breakage, further damaging DNA.
• Why is this important? Handle EtBr carefully to avoid degrading your DNA samples and harming yourself, as it’s a mutagen.
Ethidium bromide (EtBr)
- Nucleases and DNA Protection
a) Nucleases on Human Skin
• ______are enzymes that break down DNA or RNA.
• These enzymes are present naturally on human skin. If your fingers touch DNA samples or tools, the enzymes can degrade the DNA.
• Why is this important? Always use gloves and avoid touching DNA samples directly to prevent contamination or degradation.
Nucleases
• Storage of DNA in buoyant CsCl2 with_______ in rice (70)
Ethidium ramodes
• For long-term storage of DNA, it is best to store in high salt in the presence of high EDTA at____
pH 8.5
Storage for DNA
•______ is one of the best and simplest conditions for storing DNA.
•_______ causes extensive single and double strand breakage.
•–______is probably excellent for long-term storage
5 degree C
–20 C 70 C
