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Molecular Biology Lec > LESSON 3 LABORATORY SET-UP AND EQUIPMENTS > Flashcards

LESSON 3 LABORATORY SET-UP AND EQUIPMENTS Flashcards

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1
Q

Unwanted nucleic acids into the specimen

A

Contamination

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2
Q

Repeated amplification of the same target sequence leads to accumulation of amplification products (?) in the laboratory environment

A

10^9

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3
Q

Contamination Sources:

A
  1. Cross contamination between specimens
  2. Amplification product contamination
  3. Laboratory surfaces
  4. Ventilation ducts
  5. Reagents/supplies
  6. Hair, skin, saliva, and clothes of lab personnel
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4
Q

Contamination will be reduced.
1. Organization of [?]
2. Correct handling of [?]
3. Hands must be [?] and gloves and lab coats changed when moving between the designated areas.
4. Each room/area needs a separate set of [?]

A

workspace and equipment

reagents

washed

clearly labelled equipments and reagents

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5
Q

[?]Guidelines in Securing a License Operate a COVID-19 Testing Laboratory in the Philippines

A

AO 14 series 2020

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6
Q

COVID-19 testing shall only be done in a

A

DOH licensed

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7
Q

COVID-19 testing laboratory shall be a [?], with its own designated working room

A

separate unit

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8
Q

Strict adherence to biosafety and biosecurity guidelines, infection prevention and control prescribed by the [?]

A

RITM based on WHO recommendations

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9
Q

COVID-19 testing laboratories shall be strictly prohibited from [?]of examinations

A

outsourcing

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10
Q

only use [?] testing kits, reagents and devices

A

FDA registered

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11
Q

supervised by a [?], with training in Molecular Laboratory Diagnosis

A

Board Certified Clinical Pathologist

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12
Q

Physical Facilities according to AO 14 series 2020 Dedicated space for the following:

A
  1. Specimen reception
  2. Sample Preparation room/ Virus Inactivation and Nucleic acid Extraction (Pre-PCR)
  3. Reagent storage/ Handling Master mix preparation room (Pre-PCR)
  4. PCR/ Amplification
  5. Clerical activities room
  6. Template addition room
  7. Changing room
  8. Post-PCR area
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13
Q

Both personnel, including

A

cleaning personnel, and specimens

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14
Q

• Amplification [?]

A

product-free to product-rich

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15
Q

• Remove [?] before leaving one area

A

PPE

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16
Q

• Avoid or limit [?]

A

reverse direction

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17
Q

Features of each area
1. Each area has separate sets of [?]
2. [?]- serves as a clean bench area
3.  - are utilized for the aseptic transfer of materials between two controlled areas without the need for personnel movement
4. Air pressure
Reagent preparation:
Sample preparation:
Post-PCR:

A

equipment and supplies

Dead air box with UV light

Pass boxes

Positive pressure ; Negative pressure ; Negative pressure

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18
Q

01 Sample preparation/ Nucleic acid Extraction/ Virus inactivation

A

Minifuge
BSC Class II A2
Autoclave
Heating block
Cold rack

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19
Q

01 Sample preparation/ Nucleic acid Extraction/ Virus inactivation Others

A

Vortex mixer
Pipettes
Refrigerator with freezer for specimens
Refrigerator with Freezer for nucleic acid extracts
Minifuge
Computer and printer for accessioning

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20
Q

02 Master Mix

A

Filter pipette tips
Laminar flow cabinet
PCR cabinet

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21
Q

02 Master Mix Others

A

Refrigerator and freezer for reagents
Cold rack for PRC tubes
Minifuge
Microcentrifuge
Micropipettes
LFC/ PCR cabinet
Vortex mixer

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22
Q

03 Amplification/ PCR Room

A

Microplate spinner
Thermocycler
RT-PCR

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23
Q

03 Amplification/ PCR Room Others

A

Minicentrifuge
Thermocycler

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24
Q

04 Post- PCR Room

A

Electrophoresis
UV transilluminators
Gel documentation (E-box)
PCR-ELISA

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25
Q

Separation of Nucleic acid from proteins and other cellular debris

A

Minifuge

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26
Q

in extraction, DNA should be separated from an organic solvent

A

Minifuge

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27
Q
  • Protects both the personnel and product
A

BSC Class II A2

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28
Q
  • required by AO 14
A

BSC Class II A2

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29
Q
  • Steam under pressure to kill bacteria, viruses, fungi, and spores in laboratory equipments and media
A

Autoclave

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30
Q
  • 121oC; 15 psi (pressure); 15 to 30 mins
A

Autoclave

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31
Q
  • for glasswares and culture media
A

Autoclave

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32
Q
  • for extraction kits that requires heating for elution buffer
A

Heating block

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33
Q
  • some extraction kits needs to be heated
A

Heating block

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34
Q
  • for PCR tube: allows samples to stay cold longer
A

Cold rack

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35
Q

– for extraction of DNA and separation of nucleic acid (supernatant) from cellular debris (lipids) to set down at the bottom

A

Centrifugation

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36
Q

Different case for elusion

A

Centrifugation

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37
Q
  • are used to prevent cross contamination or “carry over” from one sample to another
A

Filter pipette tips

38
Q
  • to prevent carryover of the piston of the micropipette
A

Filter pipette tips

39
Q
  • Product protection only
A

Laminar flow cabinet

PCR cabinet

40
Q
  • has HEPA filter, which filters the air out
A

Laminar flow cabinet

41
Q
  • along with dead air box that does not filter the air coming from the cabinet
A

PCR cabinet

42
Q
  • to mix the microplates placed on the thermocycler
A

Microplate spinner

43
Q

RPCR machine

A

Thermocycler

44
Q

Thermocycler has varying temp

A
  1. Denaturation:94oC
  2. Annealing: 62-72o
  3. Extension: 4oC
45
Q
  • rapid result
A

RT-PCR

46
Q
  • not present in UB yet (million peso worth)
A

RT-PCR

47
Q

Not present in AO 14

A

Post- PCR Room

48
Q
  • requires good pipetting technique
A

Electrophoresis

49
Q
  • new UV transilluminator
A

Gel documentation (E-box)

50
Q
  • UV will make the fluorescence stain more visible
A

Gel documentation (E-box)

UV transilluminators

51
Q

Combined CC, IS, MolBio (Ab + enzyme)

A

PCR-ELISA

52
Q

Ultrasensitive for the detection of nucleic acid/DNA sequence

A

PCR-ELISA

53
Q

Number1 enemy in the lab

A

Contamination

54
Q

Working from clean to dirty

A

Organization of workspace and equipment

55
Q

Micropipettes have air filters

A

Correct handling of reagents

56
Q

Aerosols are created upon aspiration of reagents

A

Correct handling of reagents

57
Q

Reagents are handled in a specific area only

A

Correct handling of reagents

58
Q

Changing rooms limit the contamination

A

Hands must be washed and gloves and lab coats changed when moving between the designated areas.

59
Q

Ex.:
- Pipettes are dedicated in the PCR room only (one pipette per room)
- Minifuge are dedicated in the specimen reception only

A

Each room/area needs a separate set of clearly labelled equipments and reagents

60
Q

Filipino guideline based on WHO recommendation or the requirements to operate in a COVID-19 lab

A

AO 14 series 2020 Guidelines in Securing a License Operate a COVID-19 Testing Laboratory in the Philippines

61
Q

dedicated area where specimen is received

A
  1. Specimen reception
62
Q

Ex. For COVID-19 testing

A
  1. Sample Preparation room/ Virus Inactivation and Nucleic acid Extraction (Pre-PCR)
63
Q

Should be NO contamination (false +)

A
  1. Sample Preparation room/ Virus Inactivation and Nucleic acid Extraction (Pre-PCR)
64
Q

Anything involved with preparing reagents, aliqouting it, handling it, and

A
  1. Reagent storage/ Handling Master mix preparation room (Pre-PCR)
65
Q

Should be NO contamination (false +)

A
  1. Reagent storage/ Handling Master mix preparation room (Pre-PCR)
66
Q

Products of amplification (amplicons) should be excluded in this area

A
  1. Reagent storage/ Handling Master mix preparation room (Pre-PCR)
67
Q

PCR/ Amplification Main equipment:

A

Thermocycler and RT-PCR

68
Q

: does not photocopy DNA (convert to complementary DNA or c DNA first)

A

Thermocycler

69
Q

For correlating, analysing, understanding the results, analysing, and administrative work

A

Clerical activities room

70
Q

template DNA (extracted DNA, cDNA, plasmid) is added to the reagent

A

Template addition room

71
Q

photocopied by the RT-PCR

A

Template addition room

72
Q

where template DNA and reagent are mixed tpgether

A

Template addition room

73
Q

there could be more than 1 in a covid-19 area

A

Changing room

74
Q

not required in RT-PCR laboratory (results are already apparent)

A

Post-PCR area

75
Q

electrophoresis, PRC ELIsa detection, DNA sequencing

A

Post-PCR area

76
Q

“contaminated/dirty area”

A

Post-PCR area

77
Q

Present in UB since we have thermocycler

A

Post-PCR area

78
Q

Unidirectional – either left to right or right to left

A

Floor Plan of Free-Standing COVID-19 Testing Lab

79
Q

– must be restricted to minimum

A

Backflow traffic

80
Q

[?] (clean area; amplification prod-free) → [?] → [?] (dirty area; amplification prod-rich)

A

Master mix/Rgt prep room

Sample prep room

amplification area

81
Q

[?] goes inward, meaning the air stays there (contained)

A

negative pressure

82
Q

Air is not circulating

A

Dead Air Box

83
Q

Uses UV to sanitize the area

A

Dead Air Box

84
Q

Could be present in all rooms

A

Laminar Air Flow and Dead Air Box

85
Q

Could protect the product from any contamination, but does not
protect the personnel

A

Laminar Air Flow and Dead Air Box

86
Q

For a sterilized area to work on

A

Laminar Air Flow and Dead Air Box

87
Q

Depending on need/budget of the lab

A

Laminar Air Flow and Dead Air Box

88
Q

Ensures that the products are not contaminated

A

Laminar Air Flow and Dead Air Box

89
Q

Used for reagent mix or drying area

A

Laminar Air Flow and Dead Air Box

90
Q

are utilized for the aseptic transfer of materials between two
controlled areas without the need for personnel movement

A

Pass boxes

91
Q

Used to pass an extracted nucleic acid to a template addition to
prevent the personnel from moving around further

A

Pass boxes

Molecular Biology Lec (13 decks)

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