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Mol Bio Lab > (M) L1: PCR: Pre-Analytic and Optimization > Flashcards

(M) L1: PCR: Pre-Analytic and Optimization Flashcards

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1
Q

An in-vitro enzymatic reaction to amplify a defined DNA region that was developed during 1985

A

Polymerase Chain Reaction (PCR)

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2
Q

T or F

PCR is a vito enzymatic reaction

A

F (in-vitro enzymatic reaction)

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3
Q

One of the most useful techniques where DNA concentration increases exponentially at each cycle

A

Polymerase Chain Reaction (PCR)

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4
Q

T or F

In every PCR cycle, the amplification products become template for next rounds

A

T

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5
Q

Why is PCR one of the most useful techniques?

A

Because of its sensitivity and speed

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6
Q

Prior PCR, old methods took how long to amplify DNA?

A

Days to Weeks

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7
Q

PCR amplifies DNA for how long?

A

1-3 hours only wow

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8
Q

T or F

PCR require large amounts of DNA

A

F (PCR require small amounts of DNA)

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9
Q

What fields is PCR useful for?

A

Research, Genetics, Forensics

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10
Q

This encompasses all the procedures before specimen are measured by the analyzer

A

Pre-analytical phase

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11
Q

Give me at least three examples of pre-analytical phase (or you can use this for familiarization idk up to you)

A

test selection
patient identification
collection and handling of the sample
sorting
pipetting
centrifugation
disinfection
optimization of reagents, etc

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12
Q

T or F

Negligence in pre-analytical phase of PCR does not change the results

A

F (leads to erroneous results)

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13
Q

Is the number one enemy in the laboratory which can originate from any point in the procedure

A

Contamination

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14
Q

This is the major concern in contamination

A

Laboratory design

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15
Q

T or F

In the laboratory, when contamination occur, contaminants can also be amplified by PCR

A

T so much (even a single molecule can give rise to product)

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16
Q

What to implement to prevent contamination?

A

Contamination Control

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17
Q

Identify what potential contamination sources in pre-analytical phase:

PCR products from previous amplifications when opening PCR tubes for gel electrophoresis wherein liquid may spread through air and contaminate other tubes (cross-contamination)

A

Amplicon aerosol

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18
Q

Potential contamination sources in pre-analytical phase: amplicon aerosol

Can prevent amplicon aerosol

A

real-time PCR (since you don’t need to open tubes prior gel electrophoresis)

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19
Q

Potential contamination sources in pre-analytical phase: amplicon aerosol

Cross contamination can cause what results?

A

False-positive for initially negative samples

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20
Q

Identify what potential contamination sources in pre-analytical phase:

The actual DNA segment to be amplified is contaminated prior to the procedure

A

Target template contaminants

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21
Q

Potential contamination sources in pre-analytical phase: target template contaminants

What 2 procedures can cause target template contaminant?

A
  1. improper wearing of PPE
  2. Improper extraction of sample
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22
Q

Potential contamination sources in pre-analytical phase: target template contaminants

If PPE are worn incorrectly, what could happen to the final product?

A

Nucleic acids are incorporated

Becauseof the DNA from out body (hair, saliva etc.) nakaklabas

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23
Q

Identify what potential contamination sources in pre-analytical phase:

This involves contamination of reagents which is acquired by forgetting to cover reagents, etc.

A

Reagent contamination

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24
Q

What must be the pressure of the reagent preparations rooms?

A

Positive (outward; para walang contaminants makapasok)

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25
Q

Potential contamination sources in pre-analytical phase: reagent contamination

Once the reagent is contaminated what should you do?

A

Throw out entire container

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26
Q

Potential contamination sources in pre-analytical phase: reagent contamination

What should you do in opening containers?

A

Write the date when it was opened so next user has an idea

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27
Q

Potential contamination sources in pre-analytical phase: reagent contamination

Reagent contamination lead to what results?

A

Error (Multiple bands show up)

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28
Q

What are the 3 Potential contamination sources?

A
  1. Amplicon aerosol
  2. Target template contaminants
  3. Reagent contamination
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29
Q

What are the 7 ways to control contamination?

A
  1. Space and Time preparation
  2. Equipment in PCR Laboratory
  3. Pre-PCR and Post-PCR setup
  4. DUTP-UNG system
  5. Environmental conditions
  6. Sterilization of reagents
  7. Contamination control
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30
Q

Identify ways to control contamination:

There should be enough space and time for the procedure

A

Space and Time preparation

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31
Q

7 ways to control contamination: space and time preparation

What should you do if there is little space in laboratory?

A

Manage time and distribute tasks through different time periods

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32
Q

T or F

It is recommended that there should only be one room for every laboratory procedure

A

F (recommended to have separate rooms)

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33
Q

Identify ways to control contamination:

→ There should be gloves dedicated to pre or post procedures

→ Wherever the equipment is designated, it should stay there and not be transferred to other rooms

A

Equipment in PCR laboratory

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34
Q

7 ways to control contamination: equipment in PCR laboratory

What do you use to wipe bench tops, hoods, or any surface that was in contact with the specimen to degrade the DNA and RNA

A

10% bleach

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35
Q

7 ways to control contamination: equipment in PCR laboratory

After wiping the bench tops with 10% bleach, what should you do to avoid rust and ensure sample remnants are destroyed

A

70% ethanol

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36
Q

T or F

In crime scenes, gloves are wiped with bleach and air dried before handling the evidence

A

T

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37
Q

7 ways to control contamination: equipment in PCR laboratory

Pipettes, changing tips, isolation cabinets BSCs are subjected under what?

A

UVL decontamination treatment

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38
Q

T or F

The efficiency of UVL decontamination treatment depends on wavelength, energy, and distance

A

T

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39
Q

7 ways to control contamination: equipment in PCR laboratory

Skin and eye exposure to the UVL can cause?

A

Mutations and damage to plastics

correction:mutation to cell if for tao; damage to plastics if sa equip

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40
Q

Identify ways to control contamination:

The clean area of the pre-PCR and dirty area of post-PCR procedures should be further divided

A

Pre-PCR and Post-PCR setup

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41
Q

7 ways to control contamination: pre-PCR and post-PCR setup

Clean area of the pre-PCR procedures should be further divided into what?

A
  1. Sample processing rooms
  2. Master mix preparation rooms
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42
Q

7 ways to control contamination: pre-PCR and post-PCR setup

What 2 equipments are used for master mix preparation?

A

Box cabinet, BSCs

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43
Q

7 ways to control contamination: pre-PCR and post-PCR setup

The dirty area of the post-PCR procedures should be further divided into what?

A
  1. Amplification room
  2. Visualization room
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44
Q

T or F

Post-PCR does not need to have separate room since it does not have any effect

A

F (should be separated from other rooms due to the high concentration of DNA)

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45
Q

T or F

materials such as gloves should be present in pre-PCR only

A

F (pre and post PCR)

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46
Q

Identify ways to control contamination:

A process wherein the enzyme uracil-N glycosylase is added to the reaction/PCR mix at the beginning of each PCR

A

DUTP-UNG system

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47
Q

7 ways to control contamination: DUTP-UNG system

What is the full meaning of DUTP-UNG system?

A

“Deoxyuridine Triphosphate” and “Uracil DNA Glycosylase”

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48
Q

7 ways to control contamination: DUTP-UNG system

What enzyme is added to PCR mix at the beginning of each PCR which degrades any nucleic acid containing uracil

A

uracil-N glycosylase

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49
Q

T or F

uracil-N glycosylase is added to PCR mix to prevent carry-over contamination from previous PCR products so that no further amplification can be done

A

T

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50
Q

7 ways to control contamination: DUTP-UNG system

What is the incubation period of UNG enzyme?

A

50ºC for 2-10 minutes

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51
Q

T or F

A sign that UNG enzyme is degraded is once renaturation occurs

A

F (once denaturation occurs)

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52
Q

Identify ways to control contamination:

Positive and Negative pressured rooms in lab

A

Environmental conditions

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53
Q

7 ways to control contamination: Environmental conditions

→ This type of pressured room allow for the room to have higher pressure than the surrounding areas hence contaminants cannot enter the room

→ airflow is outward

A

Positive (kasi if palabas yung toxic na tao sa buhay mo, positive life mo)

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54
Q

7 ways to control contamination: Environmental conditions

→ This type of pressured room decrease the pressure makes the air flow inward hence no contaminants can get out

→ Usually for dirty areas

→ airflow is inward

A

Negative (kasi if pinapasok mo yung toxic na tao sa buhay mo, negative life mo

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55
Q

T or F

Negative pressure rooms are clean areas

A

F (are dirty areas because of amplicons)

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56
Q

7 ways to control contamination: Environmental conditions

Give me an example where positive pressure is used

A

Reagent preparation

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57
Q

7 ways to control contamination: Environmental conditions

Give me an example where negative pressure is used

A

DNA and RNA isolation, amplification, template-adding

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58
Q

T or F

If there is a BSC present, there is no need for pressurization except for when the sample is COVID

A

T

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59
Q

Identify ways to control contamination:

Autoclaving all materials to be used (e.g. jars, pipette tips, etc.)

A

Sterilization of reagents

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60
Q

Identify ways to control contamination:

Positive and negative controls to know and ensure accuracy of assay

A

Contamination control

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61
Q

7 ways to control contamination: contamination control

Ensures that enzymes are active, buffers are optimal, primers are priming, sequences are correct, and thermocyclers are working

A

Positive controls

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62
Q

7 ways to control contamination: contamination control

aka “contamination or reagent control”

Ensures that the reaction mix is not contaminated by template DNA or amplified products from previous runs, only the reagent is present

A

Negative controls

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63
Q

T or F

Negative controls can manifest/show result

A

F (it only checks if sample is contaminated)

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64
Q

7 ways to control contamination: contamination control

Use DNA sequences that lack the target sequence to ensure that the primers will not anneal to non-target regions (checks primer specificity)

A

Negative template controls

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65
Q

If you see this card, review the Laboratory Designs acc. to Increasing Size

A

this important so go now, read it

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66
Q

Identify the PCR Laboratory Setup:

Positive displacement pipettes, Gloves and laboratory coat, Refrigerator, Water bath or Dry heat block, Laminar flow BSC, Cell lysis reagents

A

Specimen preparation

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67
Q

Identify the PCR Laboratory Setup:

Amplification reagents and supplies, Positive displacements pipettes, Laminar flow BSC, Gloves and Laboratory coat, Water bath or Dry heat block

A

Reagent preparation (only diff with specimen preparation is wala siyang ref, cell lysis reagents)

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68
Q

Identify the PCR Laboratory Setup:

Thermal cycler, Pipettors, Detection unit (electrophoresis unit, incubator, late washer and reader water bath), Refrigerator and Freezer, Reagents and Supplies for detection

A

Amplification and Detection

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69
Q

What are the reagents for PCR that needs to be put in freshly filled ice bucket? (or use this as familiarization idk up to you)

A

Primers, DNA Polymerase, dNTPs, DNA Template, Buffer, Water

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70
Q

How are reagents organized in the workbench? (or use this as familiarization idk up to you)

A

PCR Tubes, PCR rack, Ethanol-resistant marker, Micropipettors, Thermal cycler, Master mix, Electrophoresis Apparatus

71
Q

T or F

Rigorous hand movements may cause aerosols and contaminations, so it is important to minimize it

A

T so much

72
Q

T or F

if the materials are already close by, there is more movement therefore less airborne particles will be produced

A

F (there is less movement, kasi nga close na sila)

73
Q

Placing reagents on ice prevents what?

A

Nuclease activity (that may breakdown nucleic acid functionality)

74
Q

→ Best done in closed systems
→ Protects the sample against contamination
→ Quality assessment of nucleic acid

A

Specimen Preparation

75
Q

Specimen Preparation:

In blood and bone marrow specimen what must be inhibited?

A

Clotting

76
Q

Specimen Preparation:

What anticoagulants should be used to inhibit clotting and chelates Magnesium and Calcium in sample

A

EDTA and Citrate

chelate or bind

77
Q

T or F

Magnesium and Calcium are cations that destabilize ribonuclease which help to amplify DNA or RNA, hence need to be activated)

A

F (stabilize ribonuclease which destroy DNA or RNA, hence need to be deactivated)

78
Q

Specimen Preparation:

This anticoagulant is not recommended because it inhibits PCR functions

A

Heparin

78
Q

Specimen Preparation:

What are the PCR inhibitors? (or use this card for familiarization idk up to you)

A

Heparin, Hemoglobin, Urea, Excess KCl & NaCl, Other salts, Ionic detergents, Alcohols, etc.

78
Q

Identify what factor under Specimen Preparation:

Establish the minimum acceptable yield and purity (the limits indicate good amplification)

A

Quality assessment of nucleic acid

79
Q

Specimen preparation: quality assessment of nucleic acid

What equipment is used to know amount of nucleic acid extracted?

A

Spectrophotometer

80
Q

Specimen preparation: quality assessment of nucleic acid

Deals with the presence or absence of contaminants and whether the extracted sample was pure DNA or RNA

A

Sample purity

81
Q

T or F

Consider the stability of nucleic acid and reagents

A

T so much

82
Q

This is the process of making something fully perfect, functional, or effective as possible

A

PCR Optimization

83
Q

This is the optimization of reaction conditions is done in order to obtain successful results

ex: right concentrations, temperatures, etc.

A

PCR Optimization

84
Q

This is the optimizations of various techniques vary across each one

A

PCR Optimization

85
Q

T or F

In PCR optimization, proper adjustment must be done accordingly

A

T

86
Q

What are the 4 factors affecting PCR?

A
  1. Primer design
  2. Reaction buffer and additives
  3. Denaturation, annealing, and elongation temperature and time
  4. Cycle number
87
Q

Understanding reagent functions is critical when deciding how to best alter the reaction conditions to obtain the desired product

A

Manipulating PCR Reagents

88
Q

T or F

Wrong concentrations lead to false results

A

T

89
Q

T or F

When troubleshooting the PCR, two reagent should be manipulated at a time

A

F (only one reagent should be manipulated at a time)

90
Q

Success of PCR may rely on changing the concentration of what reagents?

A

MgCl2, dNTPs, primers, template DNA, or DNA polymerase

91
Q

Are critical components of the reaction which determines the specificity of the PCR

A

Primers

92
Q

Are short single-stranded DNA fragments that anneal to the upstream and downstream regions of the DNA to be amplified

A

Primers

93
Q

What are the two orders in primers?

A

forward and reverse primers (one is for the 3’ strand while the other is for the 5’ strand)

94
Q

T or F

Primers can only be readily bought

A

F (You can also design your own primer)

95
Q

T or F

Designing your primer leads to more specificity depending on the nature of the experiment

A

T

96
Q

T or F

Primer needs to be specific to the target region

A

T

97
Q

Identify if Do’s or Dont’s in Principles of Personal Primer Design:

Primer should be 20-30 nucleotides in length

A

Do’s

98
Q

Do’s or Dont’s in Principles of Personal Primer Design:

How many nucleotides should be the length of the primer?

A

20-30 nucleotides, but 17-30 in other books

99
Q

Do’s or Dont’s in Principles of Personal Primer Design:

T or F

The longer the primer, the more specific and more efficient at hybridization

A

F (the longer the primer, the more specific but LESS efficient at hybridization)

kc mas longer mas mabagal

100
Q

Do’s or Dont’s in Principles of Personal Primer Design:

T or F

Anything less than 20 (or 17) is less/nonspecific but more efficient at hybridization

A

T

kc mas shorter mas mabilis

101
Q

Identify if Do’s or Dont’s in Principles of Personal Primer Design:

Optimal melting temperatures (Tm) range from 52-58ºC but can be expanded to 45-65ºC

A

Do’s

102
Q

Do’s or Dont’s in Principles of Personal Primer Design:

Optimum melting temperature of primers range from?

A

52-58ºC

103
Q

Do’s or Dont’s in Principles of Personal Primer Design:

Optimum melting temperature of primers range from 52-58ºC, but can be expanded to what temperature range?

A

45-65ºC

104
Q

Do’s or Dont’s in Principles of Personal Primer Design:

T or F

Optimal melting temperatures are dependent on A=T bonds

A

F (G≡C bonds, as they have more bonds)

105
Q

Do’s or Dont’s in Principles of Personal Primer Design:

Stronger bonds indicate what in melting temperature?

A

indicate increased melting temperature (stronger bonds such as G=C)

106
Q

Do’s or Dont’s in Principles of Personal Primer Design:

Optimal G≡C content should range between?

A

40-60%

107
Q

Do’s or Dont’s in Principles of Personal Primer Design:

3’ ends should contain what in order to clamp the primers and prevent “breathing” of ends therefore increasing efficiency

A

G or C

108
Q

Identify if Do’s or Dont’s in Principles of Personal Primer Design:

Secondary structures (hairpins) within each primer

A

Don’ts

109
Q

Do’s or Dont’s in Principles of Personal Primer Design:

T or F

Hairpin loops cause potential dimerization

A

T

110
Q

Do’s or Dont’s in Principles of Personal Primer Design:

These prevent annealing to the target DNA and will instead anneal to itself

A

Hairpin loops

111
Q

Identify if Do’s or Dont’s in Principles of Personal Primer Design:

Dinucleotide repeats/annealing withself

A

Dont’s

112
Q

Do’s or Dont’s in Principles of Personal Primer Design:

These cause slipping along the primed segment or hairpin loops

A

Dinucleotide repeats/annealing withself

113
Q

Do’s or Dont’s in Principles of Personal Primer Design:

Can cause both primers to bind with each other instead of the target DNA (primer dimer)

A

Single base runs

114
Q

Do’s or Dont’s in Principles of Personal Primer Design:

Consists of two primer molecules that have attached (hybridized) to each other because of strings of complementary bases in the primers.

A

Primer dimer, btw google meaning lang to

115
Q

Increases primer concentration leads to what?

or TOO HIGH PRIMER CONC.

s

A

Non-specific products and primer dimers

116
Q

What primer concentration are ideal based on the photo that ma’am showed?

A

0.1 or 0.25 because there are no shadow bands present

117
Q

Magnesium Concentration:

Optimal concentration for Taq DNA Polymerase

A

1.5 - 2.0 mM

118
Q

Magnesium Concentration: (TOF)

(TOF). The final concentration of Mg2+ is
0.05 up to 0.5 mM

A

False kween (0.5-5.0 mM)

119
Q

Magnesium Concentration: (TOF)

(TOF). The optimal concentration depends on template, buffer, DNA, and dNTPs.

A

T

120
Q

Required co-factor for thermostable DNA polymerase and important for successful amplification

A

Magnesium

121
Q

Magnesium Concentration:

(TOF) DNA polymerase will stop extending the primer without [Mg2+]

A

F (can still extend but not that active w/out Mg2+)

122
Q

Magnesium Concentration: (TOF)

Why would there be no/less PCR products seen if the [Mg2+] concentration is low?

A

kc bc due to the reduced polymerase activity

(remember that w/out or less Mg2+, tamad c DNA polymerase hence the reduced products)

123
Q

Magnesium Concentration: (TOF)

Too much or higher concentration of [Mg2+] yields better PCR products

A

F (higher [Mg2+] results in undesired products)

nag ↑ sensitivity = continuous amplification → decreased specificity

124
Q

Typical concentration of each piece of Deoxynucleotides (dNTPs)

A

200 µM

125
Q

Building blocks of new DNA strands

A

dNTPs

126
Q

Deoxynucleotides (dNTPs): TOF

Lower concentrations of dNTPs (50-100 µM)
enhance both the specificity and fidelity of the
reaction, but reduces yield

A

T

127
Q

Deoxynucleotides (dNTPs): TOF

Higher concentrations of dNTPs increase
yields particularly in long PCR but can reduce
fidelity and can inhibit PCR

A

T tlga

128
Q

First invented/discovered/performed PCR by using DNA polymerase isolated from e.coli

A

Kary Mullis

129
Q

What do you call the enzyme derived from a thermophilic bacterium

A

Taq Polymerase

Thermus (genus); Aquaticus (species) [aq ay deadicus na]

130
Q

Thermostable DNA polymerase: (TOF)

With the taq polymerase, DNA pol can now be added once in the beginning of the procedure but will degrade after the heating and cooling cycle

A

FALSE, HINDI! (MAS STABLE NA NGA DAPAT SO MAINTAINED ACTIVITY THROUGHOUT THE PROCEDURE!!)

dna poly can now resist higher temp hehe

131
Q

Thermostable DNA polymerase:

👹Enumerate👹 other enzymes aside from Taq

A

Thermus thermophilus
(Tth) and Pfu

pfu not mentioned in lab lec but nasa trans so okay?

132
Q

Thermostable DNA polymerase: TOF

If the concentration of the DNA polymerase is
low = less, insufficient, or no products

A

T

ket marami iyong dNTPs, kung less ang dna poly insufficient ang ma-elongate ng primer

133
Q

Thermostable DNA polymerase: TOF

Too much DNA polymerase will increase the SPECIFICITY

A

F (decrease)

too much polymerase increases the synthesis of nonspecific pcr products

134
Q

Thermostable DNA polymerase:

In a typical 50 µL reaction, what is the ideal unit of DNA polymerase to amplify your target DNA?

A

1 - 2 units

HOWEVER, it may be necessary to adjust the enzyme amt…

… with difficult templates

135
Q

Thermostable DNA polymerase

In an experiment with inhibitors (heparin, NaCl, etc.) present in the DNA sample, what would be your course of action to optimize PCR yield?

A

Increase the amount of DNA polymerase

HOWEVER,nonspecific products may appear with higher enzyme concentration

136
Q

TOF

The template DNA could be complementary DNA, genomic DNA, or plasmid DNA

A

T

137
Q

TOF

Optimal template amount varies based on the type of DNA polymerase used

A

T

138
Q

Template DNA: (TOF)

With regards to the primer ratio, primers would be able to find their complimentary sequence even if there is too little template

A

F

139
Q

Template DNA

Too much template over the primer would result to what?

A

Mispriming and may also inhibit the polymerase

140
Q

Template DNA

Normally, the target sequences to be amplified should be kept at how many kilobases

A

< 3 kb

LESS THAN HINDI PUSO

141
Q

Template DNA: (TOF)

Fragments up to 10 kilobases cannot be amplified

A

F (pwede but longer time to amplify)

142
Q

Template DNA: (TOF)

You should always check the quality and purity of your DNA templates before amplification

A

T, yes naman be competent, dear RMT

143
Q

PCRs are carried out in a ________ that will provide a suitable chemical environment for the DNA polymerase

A

Buffer babe, buffer.

144
Q

Buffer: (TOF)

It is important to follow buffer recommendations by the enzyme’s supplier, since the optimal PCR buffer is dependent upon the DNA polymerase used.

A

chrew

145
Q

Buffer pH usually ranges from

A

8 - 9.5

146
Q

Buffer

Why is there a need to add chemical additives or co-solvents in the buffer

A
  • It helps improve the amplification specificity by reducing the mispriming
  • Enhance amplification efficiency by removing secondary structures
147
Q

Buffers (TOF)

Chemical additives or co-solvents above their optimum concentration does not have an effect on PCR

A

F (it can inhibit the PCR, above nga ng optimum eh)

148
Q

Recommended initial denaturation temp and duration prior to PCR cycling to fully denature the DNA

A

90 - 96°C for 2 minutes

*varies depending on source but yan ang ifollow-

95° C on ppt, but can be expanded to 90-96° C

149
Q

Denaturation Temp and Duration: (TOF)

We must always incubate DNA in longer and higher temps

A

F (avoid longer or higher temp)

denaturation lang to hindi ka naman magluluto ng nilaga

150
Q

Denaturation Temp and Duration:

What will happen if the temperature and duration is too high/long?

A

increase of sensitivity

*possibility of decreasing taq polymerase acitivity

151
Q

Denaturation Temp and Duration

What will then happen if the temperature and duration is too low?

A

incomplete denaturation and low amplification

152
Q

Denaturation Temp and Duration:

During thermocycling what is the typical temp and duration for denaturation?

A

15-30 seconds denaturation at 95°C

153
Q

Annealing Temp and Duration

Typical annealing temperature falls in the range of?

A

50 - 60° C

that is 5° C BELOW the lowest primer’s Tm

154
Q

Annealing Temperature and duration: (TOF)

The success of the PCR does not depend on maintaining a high ratio of specific to non-specific annealing of the primer molecules

A

F

155
Q

When you see multiple bands/shadows in electrophoresis what will you adjust to merge the banding?

A

Annealing temperature

156
Q

Annealing Temperature and Duration

What is the difference between annealing and melting temperature?

A

Annealing temp - process where primer binds to template

Melting temp - temp at which half of the oligonucleotide molecules are single stranded and half are double standed

157
Q

Why shouldn’t the Annealing Temperature be equal to the Tm?

A

annealing temp must be low enough to enable hybridization and high enough to prevent mismatched hybrids from forming

158
Q

Annealing temperature and Duration: (TOF)

Annealing temperature determines the hybridization between the primer and polymerase

A

F (primer and template)

159
Q

Annealing Temperature and Duration

What should you check before setting the annealing temperature?

A

Melting Temperature

160
Q

Annealing Temp and Duration:

If the melting temperature is 55°C, what would be the annealing temp?

A

50° C (basta daw 5°C below ng Melting temp)

161
Q

Annealing Temperature and Duration:

If the melting temp is not given, what should you do?
a. omiyak
b. sisihin si bongbong
c. magcompute
D. tomalon sa jip

b (real)

A

SIKE YOU C. COMPUTE

HERE FORMULA: Tm = [4 x (G+C)] + [2 x (A + T)] °C

162
Q

Annealing Temp and Duration:

This card be reminder that thou shouldst aral the given formula for melting temp

A

amen

163
Q

Annealing Temperature and Duration: (TOF)

More G-C content on the primer = lower melting temp

A

F (higher melting temp)

164
Q

Annealing Temp and Duration: (TOF)

If annealing temp is too low, then it could lead to nonspecific amplification

A

T

165
Q

Annealing temperature and duration:

If annealing temp is too high what would be the result?

A

no amplification

primers c0nt bind to the template cuz it be denatured

166
Q

Annealing temp and duration:

What is the typical duration for annealing?

A

15-30 seconds

167
Q

Extension time

Generally, how long would be the extension time per 1000 base pairs?

A

One minute

168
Q

Extension time

what would be the extension time for a 5 kb product?

A

5 minutes

1 kb = 1000 bp

169
Q

Extension time:

For products less than 1 kb, how long is the extension time?

A

45-60 seconds

170
Q

Extension time: (TOF)

Longer extension time will increase specificity
and number of cycles, however, will give
nonspecific products

A

F (sensitivity)

171
Q

Extension time:

If you’ve set your cycles lower/fewer what does that say about your template concentration?

A

template concentration = high

172
Q
A

Mol Bio Lab (12 decks)

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