lab set up Flashcards
3 areas in the spatial separation of pre and post amplification of work areas
- sample preparation r
- reagent preparation r
- amplification r
air pressure in each areas
reagent prep - positive
sample prep - negative
post-amplification - negative
single entrance, reagent used for amplification should not be exposed to other areas.
what area is this specifically?
reagent prep
alternative to spatial separation
- class II biological safety cabinet
- dedicated areas for each work phase
- unidirectional
- automated specimen processing station
other lab design to be considered
- temperature and humidity requirements
- exhaust ventilation
- water quality
- electric outlet
- back-up power system
- eyewash
- ergonomic assessment
laboratory practices
- use positive displacement pipettes and disposable filtered pipette tips
- avoid production of aerosols when pipetting
- use of sterilized single use plasticware
- use of cleanroom sticky floor mats
- minimizes the risk of amplicon carry-over on clothing, hair, and skin
- clean punches between samples
- use of nuclease-free or autoclaved water
- aliquot oligonucleotides
- always include a blank control to check contamination
- use of electronic data system
- wipe test (swab test)
this test is to detect, localize, and remove contamination.
to identify the source of the contamination.
this is done monthly
wipe test (swab test)
cleaning materials for PCR station
- UV light
- 70% ethanol
- 10% sodium hypochlorite
- DNA away
substitution of uracil for thymine during PCR amplification
enzymatic inactivation with uracil-N-glycosylase
cleaning of chemical and enzymatic controls
- work stations should be cleaned with 10% sodium hypochlorite
- ultra-violet light irradiation
- enzymatic inactivation with uracil-N-glycosylase
false amplification potential causes
- non-optimized assay conditions
- unknown polymorphisms in target sites
- oligonucleotide concentrations too high
- nucleic acid cross-contamination
in labeling reagents, it should have __
- lot no.
- expiration sate
- storage requirement
- date of use/disposal
enumerate the use of molbio lab
- health
- medicine
- forensics
- pharmaceutical industry
- biological warfare
- drug discovery
- PCR based technology
- fluorescence in situ hybridization (FISH)
- biochip
- peptide nucleic acid (PNA)
- proteomic technology
- electrochemical detection of DNA
contamination may cause
- incorrect results
- require extensive cleanup
- loss of creditability
- impact on financial and performance
enumerate the importance of accurate pipetting
- quantitative precision
- reproducibility
- data quality
- resource efficiency
false amplification potential causes
- non-optimized assay conditions
- unknown polymorphisms in target sites
- oligonucleotide concentrations too high
- nucleic acid cross contamination
types of pippets in a molbio lab
- micropipette
- multichannel pipettes
- serological pipettes
sizes of micropipettes that we used
P10 (0.5-10uL)
P20 (2-20uL)
P100 (10-100uL)
P1000 (100-1000 uL)
micropipettes are essential for?
- PCR
- DNA quantification
- sample transfers
these pipets allow for simultaneous pipetting of multiple sample, they are commonly used in high-throughput applications
multichannel pipets
these pipets are used for transferring larger volumes, typically in milliliter range.
serological p
serological p are often used for?
- media preparation
- cell culture
- larger-scale molbio experiments
enumerate the pipetting techniques (10)
- calibration
- pipette selection
- pre-rinse
- aspiration and dispensation
- tip ejection
- avoid air bubbles
- changing tips
- vertical pipetting
- touch-off technique
- sample mixing
proper way to avoid cross-contamination when pipetting multiple samples
always change tips between samples
which is more accurate? forward or reverse?
forward
it is the preferred method when exact volumes are critical such as qPCR or DNA quantification
forward p
reverse pipetting is used for?
working with viscous or volatile fluids
principles of forward pippeting
- accurate
- preventing contamination
principles of reverse p
- minimizing viscosity and volatility effects
- reduced error
this technique reduces the risk of over-pipetting and ensures that the intended volume is accurately delivered
reverse p
pipet techniques used for qPCR, spectrophotometry, DNA quantification. also suitable for general sample transfers and dilutions
forward pipetting
this technique is ideal for transferring concentrated glycerol stocks or volatile solutions which can evaporate during pipetting
reverse
often used in the preparation for DNA/RNA extractions
pH measurement in molbio is essential to maintain?
accuracy and reproducibility
neutral pH:
acidic pH:
alkaline pH:
neutral: 7
acidic: <7
alkaline: >7
accurate pH measurement is used for?
- buffer preparation
- enzymatic reactions
- cell culture maintenance
components of a pH meter
- electrode
- meter/analyzer
- buffer solutions
- reference electrode
core component of the pH meter which contains a glass membrane sensitive to pH changes
electrode
pH values of buffer solutions
- 4.01
- 7.00
- 10.01
buffer solutions of ph meter is used for?
calibration
proper usage of a ph meter includes (8)
- calibration
- electrode conditioning
- sample preparation
- rinse between measurements
- temperature correction
- electrode maintenance
- avoid contamination
- interference
when to perform calibration?
before each series of measurements or when using a diff electrode
this helps to stabilize the electrode and maintain its performance
electrode conditioning
what sol’n to use when rinsing the electrode?
deionized water
why is it important to maintain proper records of pH measurements?
to ensure data integrity and traceability
product of PCR
amplicons
for cheap lab that cannot afford spatial separation?
separation of pre-PCR to post-PCR
purpose of spatial room
to avoid contamination
in spectrometry, ____ means contaminated
presence of bands
enumerate the lab equipments for molbio that we have in the laboratory
- gel electrophoresis
- PCR/thermal cycler
- microcentrifuge
- micropipette/serological pipet
- pH meter
- dry heat blocks
- class II biosafety cabinet
- refrigerator
used to separate mixtures of DNA, RNA, or proteins according to molecular size
gel electrophoresis
used to amplify target nucleic acid sequences into millions of copies
PCR
used to heat or cool the samples in a controlled manner
dry heat blocks
serves as a clean bench area
dead airbox with UV light
proficiency testing is performed __
twice a year
enumerate the potential sources of contamination (6)
- cross-contamination between specimens
- amplification product contamination
- laboratory surfaces
- ventilation ducts
- reagents/supplies
- hair, skin, saliva, and cloths
enumerate the equipments needed in nucleic acid extraction area (9)
- high speed centrifuge
- vortex
- pipettes
- autoclave
- purwater systems
- refrigerator
- air-conditioner
- sink
- biosafety cabinet
enumerate the equipments needed in the reagent prep room
- pipettes
- vortex
- spindown centrifuge
- refrigerator
- freezer
- laminar hood
reagent prep room should have positive pressure
enumerate the equipments needed in the PCR amplification room
- thermocycler
- UPS and voltage stabilizer
- pipette
- spindown centrifuge
- air-conditioner
enumerate the equipments needed for the analysis of PCR products
- gel eelctrophoresis room
- pipette
- electrophoresis apparatus
- balance
- microwave oven
- sink
- dark room
- UV transilluminator camera
specimens should not be exposed to what areas?
post-amplification area
enumerate the quality indicators (6)
- turnaround time
- % of failed runs
- population medium
- calibrator parameters
- graph (to identify trend or shift)
- monitor frequency and acceptable range
critical molecular assay components
enumerate under nucleic acids (5)
- primers and probes
- dNTPs
- genomic DNA
- 4 - 8C
- -15 - 25C
prepare aliqouts appropriate to workflow to limit freeze-thaw cycles
critical molecular assay components
for enzymes, what is recommended?
benchtop coolers
critical molecular assay components
enumerate the 2 in fluorescent reporters
- limit exposure to light
- amber storage tubes or wrap in foil
other QA/QC considerations (8)
- specimen storage
- lab cleanliness and waste disposal
- instrument maintenance and calibration
- instrument/method comparison
- document management
- personnel training and competency
- periodic review of QA/QC
- COOP plan
this addresses emergencies from an all-hazards approach.
COOP plan
when is a validation/verification study required?
- introduce a new testing system
- an analyte added to a test system
- a modification to a test system
- determine the analytic performance of an assay
