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

LESSON 3 LABORATORY SET-UP AND EQUIPMENTS Flashcards

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
Separation of Nucleic acid from proteins and other cellular debris
Minifuge
26
in extraction, DNA should be separated from an organic solvent
Minifuge
27
- Protects both the personnel and product
BSC Class II A2
28
- required by AO 14
BSC Class II A2
29
- Steam under pressure to kill bacteria, viruses, fungi, and spores in laboratory equipments and media
Autoclave
30
- 121oC; 15 psi (pressure); 15 to 30 mins
Autoclave
31
- for glasswares and culture media
Autoclave
32
- for extraction kits that requires heating for elution buffer
Heating block
33
- some extraction kits needs to be heated
Heating block
34
- for PCR tube: allows samples to stay cold longer
Cold rack
35
– for extraction of DNA and separation of nucleic acid (supernatant) from cellular debris (lipids) to set down at the bottom
Centrifugation
36
Different case for elusion
Centrifugation
37
- are used to prevent cross contamination or "carry over" from one sample to another
Filter pipette tips
38
- to prevent carryover of the piston of the micropipette
Filter pipette tips
39
- Product protection only
Laminar flow cabinet PCR cabinet
40
- has HEPA filter, which filters the air out
Laminar flow cabinet
41
- along with dead air box that does not filter the air coming from the cabinet
PCR cabinet
42
- to mix the microplates placed on the thermocycler
Microplate spinner
43
RPCR machine
Thermocycler
44
Thermocycler has varying temp
1. Denaturation:94oC 2. Annealing: 62-72o 3. Extension: 4oC
45
- rapid result
RT-PCR
46
- not present in UB yet (million peso worth)
RT-PCR
47
Not present in AO 14
Post- PCR Room
48
- requires good pipetting technique
Electrophoresis
49
- new UV transilluminator
Gel documentation (E-box)
50
- UV will make the fluorescence stain more visible
Gel documentation (E-box) UV transilluminators
51
Combined CC, IS, MolBio (Ab + enzyme)
PCR-ELISA
52
Ultrasensitive for the detection of nucleic acid/DNA sequence
PCR-ELISA
53
Number1 enemy in the lab
Contamination
54
Working from clean to dirty
Organization of workspace and equipment
55
Micropipettes have air filters
Correct handling of reagents
56
Aerosols are created upon aspiration of reagents
Correct handling of reagents
57
Reagents are handled in a specific area only
Correct handling of reagents
58
Changing rooms limit the contamination
Hands must be washed and gloves and lab coats changed when moving between the designated areas.
59
Ex.: - Pipettes are dedicated in the PCR room only (one pipette per room) - Minifuge are dedicated in the specimen reception only
Each room/area needs a separate set of clearly labelled equipments and reagents
60
Filipino guideline based on WHO recommendation or the requirements to operate in a COVID-19 lab
AO 14 series 2020 Guidelines in Securing a License Operate a COVID-19 Testing Laboratory in the Philippines
61
dedicated area where specimen is received
1. Specimen reception
62
Ex. For COVID-19 testing
2. Sample Preparation room/ Virus Inactivation and Nucleic acid Extraction (Pre-PCR)
63
Should be NO contamination (false +)
2. Sample Preparation room/ Virus Inactivation and Nucleic acid Extraction (Pre-PCR)
64
Anything involved with preparing reagents, aliqouting it, handling it, and
3. Reagent storage/ Handling Master mix preparation room (Pre-PCR)
65
Should be NO contamination (false +)
3. Reagent storage/ Handling Master mix preparation room (Pre-PCR)
66
Products of amplification (amplicons) should be excluded in this area
3. Reagent storage/ Handling Master mix preparation room (Pre-PCR)
67
PCR/ Amplification Main equipment:
Thermocycler and RT-PCR
68
: does not photocopy DNA (convert to complementary DNA or c DNA first)
Thermocycler
69
For correlating, analysing, understanding the results, analysing, and administrative work
Clerical activities room
70
template DNA (extracted DNA, cDNA, plasmid) is added to the reagent
Template addition room
71
photocopied by the RT-PCR
Template addition room
72
where template DNA and reagent are mixed tpgether
Template addition room
73
there could be more than 1 in a covid-19 area
Changing room
74
not required in RT-PCR laboratory (results are already apparent)
Post-PCR area
75
electrophoresis, PRC ELIsa detection, DNA sequencing
Post-PCR area
76
"contaminated/dirty area"
Post-PCR area
77
Present in UB since we have thermocycler
Post-PCR area
78
Unidirectional – either left to right or right to left
Floor Plan of Free-Standing COVID-19 Testing Lab
79
– must be restricted to minimum
Backflow traffic
80
[?] (clean area; amplification prod-free) → [?] → [?] (dirty area; amplification prod-rich)
Master mix/Rgt prep room Sample prep room amplification area
81
[?] goes inward, meaning the air stays there (contained)
negative pressure
82
Air is not circulating
Dead Air Box
83
Uses UV to sanitize the area
Dead Air Box
84
Could be present in all rooms
Laminar Air Flow and Dead Air Box
85
Could protect the product from any contamination, but does not protect the personnel
Laminar Air Flow and Dead Air Box
86
For a sterilized area to work on
Laminar Air Flow and Dead Air Box
87
Depending on need/budget of the lab
Laminar Air Flow and Dead Air Box
88
Ensures that the products are not contaminated
Laminar Air Flow and Dead Air Box
89
Used for reagent mix or drying area
Laminar Air Flow and Dead Air Box
90
are utilized for the aseptic transfer of materials between two controlled areas without the need for personnel movement
Pass boxes
91
Used to pass an extracted nucleic acid to a template addition to prevent the personnel from moving around further
Pass boxes

Molecular Biology Lec (13 decks)

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