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Molbio. > lab set up > Flashcards

lab set up Flashcards

1
Q

3 areas in the spatial separation of pre and post amplification of work areas

A
  • sample preparation r
  • reagent preparation r
  • amplification r
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2
Q

air pressure in each areas

A

reagent prep - positive
sample prep - negative
post-amplification - negative

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3
Q

single entrance, reagent used for amplification should not be exposed to other areas.

what area is this specifically?

A

reagent prep

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4
Q

alternative to spatial separation

A
  • class II biological safety cabinet
  • dedicated areas for each work phase
  • unidirectional
  • automated specimen processing station
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5
Q

other lab design to be considered

A
  • temperature and humidity requirements
  • exhaust ventilation
  • water quality
  • electric outlet
  • back-up power system
  • eyewash
  • ergonomic assessment
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6
Q

laboratory practices

A
  • 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)
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7
Q

this test is to detect, localize, and remove contamination.
to identify the source of the contamination.

this is done monthly

A

wipe test (swab test)

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8
Q

cleaning materials for PCR station

A
  • UV light
  • 70% ethanol
  • 10% sodium hypochlorite
  • DNA away
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9
Q

substitution of uracil for thymine during PCR amplification

A

enzymatic inactivation with uracil-N-glycosylase

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10
Q

cleaning of chemical and enzymatic controls

A
  • work stations should be cleaned with 10% sodium hypochlorite
  • ultra-violet light irradiation
  • enzymatic inactivation with uracil-N-glycosylase
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11
Q

false amplification potential causes

A
  • non-optimized assay conditions
  • unknown polymorphisms in target sites
  • oligonucleotide concentrations too high
  • nucleic acid cross-contamination
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12
Q

in labeling reagents, it should have __

A
  • lot no.
  • expiration sate
  • storage requirement
  • date of use/disposal
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13
Q

enumerate the use of molbio lab

A
  • 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
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14
Q

contamination may cause

A
  • incorrect results
  • require extensive cleanup
  • loss of creditability
  • impact on financial and performance
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15
Q

enumerate the importance of accurate pipetting

A
  • quantitative precision
  • reproducibility
  • data quality
  • resource efficiency
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16
Q

false amplification potential causes

A
  • non-optimized assay conditions
  • unknown polymorphisms in target sites
  • oligonucleotide concentrations too high
  • nucleic acid cross contamination
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17
Q

types of pippets in a molbio lab

A
  • micropipette
  • multichannel pipettes
  • serological pipettes
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18
Q

sizes of micropipettes that we used

A

P10 (0.5-10uL)
P20 (2-20uL)
P100 (10-100uL)
P1000 (100-1000 uL)

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19
Q

micropipettes are essential for?

A
  • PCR
  • DNA quantification
  • sample transfers
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20
Q

these pipets allow for simultaneous pipetting of multiple sample, they are commonly used in high-throughput applications

A

multichannel pipets

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21
Q

these pipets are used for transferring larger volumes, typically in milliliter range.

A

serological p

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22
Q

serological p are often used for?

A
  • media preparation
  • cell culture
  • larger-scale molbio experiments
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23
Q

enumerate the pipetting techniques (10)

A
  • calibration
  • pipette selection
  • pre-rinse
  • aspiration and dispensation
  • tip ejection
  • avoid air bubbles
  • changing tips
  • vertical pipetting
  • touch-off technique
  • sample mixing
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24
Q

proper way to avoid cross-contamination when pipetting multiple samples

A

always change tips between samples

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25
Q

which is more accurate? forward or reverse?

A

forward

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26
Q

it is the preferred method when exact volumes are critical such as qPCR or DNA quantification

A

forward p

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27
Q

reverse pipetting is used for?

A

working with viscous or volatile fluids

28
Q

principles of forward pippeting

A
  • accurate
  • preventing contamination
29
Q

principles of reverse p

A
  • minimizing viscosity and volatility effects
  • reduced error
30
Q

this technique reduces the risk of over-pipetting and ensures that the intended volume is accurately delivered

A

reverse p

31
Q

pipet techniques used for qPCR, spectrophotometry, DNA quantification. also suitable for general sample transfers and dilutions

A

forward pipetting

32
Q

this technique is ideal for transferring concentrated glycerol stocks or volatile solutions which can evaporate during pipetting

A

reverse

often used in the preparation for DNA/RNA extractions

33
Q

pH measurement in molbio is essential to maintain?

A

accuracy and reproducibility

34
Q

neutral pH:
acidic pH:
alkaline pH:

A

neutral: 7
acidic: <7
alkaline: >7

35
Q

accurate pH measurement is used for?

A
  • buffer preparation
  • enzymatic reactions
  • cell culture maintenance
36
Q

components of a pH meter

A
  • electrode
  • meter/analyzer
  • buffer solutions
  • reference electrode
37
Q

core component of the pH meter which contains a glass membrane sensitive to pH changes

A

electrode

38
Q

pH values of buffer solutions

A
  • 4.01
  • 7.00
  • 10.01
39
Q

buffer solutions of ph meter is used for?

A

calibration

40
Q

proper usage of a ph meter includes (8)

A
  • calibration
  • electrode conditioning
  • sample preparation
  • rinse between measurements
  • temperature correction
  • electrode maintenance
  • avoid contamination
  • interference
41
Q

when to perform calibration?

A

before each series of measurements or when using a diff electrode

42
Q

this helps to stabilize the electrode and maintain its performance

A

electrode conditioning

43
Q

what sol’n to use when rinsing the electrode?

A

deionized water

44
Q

why is it important to maintain proper records of pH measurements?

A

to ensure data integrity and traceability

45
Q

product of PCR

A

amplicons

46
Q

for cheap lab that cannot afford spatial separation?

A

separation of pre-PCR to post-PCR

47
Q

purpose of spatial room

A

to avoid contamination

48
Q

in spectrometry, ____ means contaminated

A

presence of bands

49
Q

enumerate the lab equipments for molbio that we have in the laboratory

A
  • gel electrophoresis
  • PCR/thermal cycler
  • microcentrifuge
  • micropipette/serological pipet
  • pH meter
  • dry heat blocks
  • class II biosafety cabinet
  • refrigerator
50
Q

used to separate mixtures of DNA, RNA, or proteins according to molecular size

A

gel electrophoresis

51
Q

used to amplify target nucleic acid sequences into millions of copies

A

PCR

52
Q

used to heat or cool the samples in a controlled manner

A

dry heat blocks

53
Q

serves as a clean bench area

A

dead airbox with UV light

54
Q

proficiency testing is performed __

A

twice a year

55
Q

enumerate the potential sources of contamination (6)

A
  • cross-contamination between specimens
  • amplification product contamination
  • laboratory surfaces
  • ventilation ducts
  • reagents/supplies
  • hair, skin, saliva, and cloths
56
Q

enumerate the equipments needed in nucleic acid extraction area (9)

A
  • high speed centrifuge
  • vortex
  • pipettes
  • autoclave
  • purwater systems
  • refrigerator
  • air-conditioner
  • sink
  • biosafety cabinet
57
Q

enumerate the equipments needed in the reagent prep room

A
  • pipettes
  • vortex
  • spindown centrifuge
  • refrigerator
  • freezer
  • laminar hood

reagent prep room should have positive pressure

58
Q

enumerate the equipments needed in the PCR amplification room

A
  • thermocycler
  • UPS and voltage stabilizer
  • pipette
  • spindown centrifuge
  • air-conditioner
59
Q

enumerate the equipments needed for the analysis of PCR products

A
  • gel eelctrophoresis room
  • pipette
  • electrophoresis apparatus
  • balance
  • microwave oven
  • sink
  • dark room
  • UV transilluminator camera
60
Q

specimens should not be exposed to what areas?

A

post-amplification area

61
Q

enumerate the quality indicators (6)

A
  1. turnaround time
  2. % of failed runs
  3. population medium
  4. calibrator parameters
  5. graph (to identify trend or shift)
  6. monitor frequency and acceptable range
62
Q

critical molecular assay components

enumerate under nucleic acids (5)

A
  • primers and probes
  • dNTPs
  • genomic DNA
  • 4 - 8C
  • -15 - 25C

prepare aliqouts appropriate to workflow to limit freeze-thaw cycles

63
Q

critical molecular assay components

for enzymes, what is recommended?

A

benchtop coolers

64
Q

critical molecular assay components

enumerate the 2 in fluorescent reporters

A
  1. limit exposure to light
  2. amber storage tubes or wrap in foil
65
Q

other QA/QC considerations (8)

A
  • 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
66
Q

this addresses emergencies from an all-hazards approach.

A

COOP plan

67
Q

when is a validation/verification study required?

A
  1. introduce a new testing system
  2. an analyte added to a test system
  3. a modification to a test system
  4. determine the analytic performance of an assay

Molbio. (6 decks)

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