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BIO 142 > Lab Final > Flashcards

Lab Final Flashcards

1
Q

What must be included in a lab notebook?

A 
Title
Date
Purpose
Methods
Data 
Conclusions
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2
Q

C. elegan control

A

heat killed S. Marcescens

cannot infect C. elegans, but smells good to them

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3
Q

C. elegans experimental plate

A

Serratia on one side

E. coli on the other

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4
Q

What should be labeled on a plate for c. elegans?

A 
Date 
Group name
Type of plate (evolution or control)
Lab section
Lab room number
Passage number
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5
Q

Parts of a scientific abstract

A

Introduction
Materials and Methods
Findings/results
Discussion

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6
Q

Components of the introduction of a scientific abstract

A

Context, knowledge gap, and objectives

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7
Q

Where does the microbiome come from?

A

Fetus is probably sterile

Bacteria is acquired at birth, changes as the human develops/changes

Bacterial composition is highly variable

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8
Q

What is the microbiome thought to be determined by?

A

Diet
Genetics/immune system
environment

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9
Q

How can the microbiome negatively affect a person’s health?

A

certain species have been identified in gastrointensional distress, weight gain, and neurological diseases

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10
Q

Is most bacteria in the microbiome beneficial?

A

yes

helps with food digestion, wound healing, gut barrier function

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11
Q

Why is the bean beetle a good model organism?

A

easy to maintain

short life cycle (about 1 month)

tiny, but not too small

eats one bean that larvae was on and never eats again…can completely control diet

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12
Q

Broad and specific research questions of bean beetle project

A

Broad: How does diet affect the microbiome?

General: Do beetles raised on different bean types have different microbiomes?

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13
Q

PEA plate

A

selectively cultivates Gram positive microbes

P = Positive

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14
Q

EMB plate

A

selectively cultivates Gram negative microbes

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15
Q

Cellulose plates

A

contains indicator that will show color change around microbes that can digest cellulose

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16
Q

Gram negative bacteria

A

have an outer membrane ontop of their peptidoglycan cell wall

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17
Q

Why do we sterilize the bean beetles?

A

sterilize their outside, so we only culture internal bacteria

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18
Q

Why do we plate both diluted and undiluted beetle microbiomes?

A

Some beetles have tons of microbes while others have much less

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19
Q

What should be labeled on the bean beetle plates?

A 
Group name
date
lab section 
lab room
beetle type
beetle #
dilution
type of plate
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20
Q

What are the 2 ways that scientists think about diversity?

A
  1. Species richness

2. Species evenness

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21
Q

Species richness

A

total number of species present

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22
Q

Species evenness

A

the numbers of each species are pretty even

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23
Q

Inverse Simpson’s Index

A

1/ (# of species A /total)^2 + (# of species B/total)^2…

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24
Q

What plates do we use for diversity measures?

A

EMB and PEA plates

since they show gram-negative vs. gram-positive bacteria

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25
Q

Morphological data on bacterial colonies

A
  1. Color
  2. Gloss
  3. Colony size
  4. Shape
  5. Elevation
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26
Q

How to determine what part of genome to sequence?

A

1) region needs to be present in all microbes
2) needs to have variable regions to identify different species
3) needs to have conserved regions to design a PCR primer

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27
Q

Why is the 16s rRNA gene commonly sequenced in bacterial DNA?

A

the 16s gene is conserved across all bacterial species and has conserved and variable regions

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28
Q

How do we amplify DNA for sequencing?

A

Polymerase Chain Reaction (PCR)

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29
Q

PCR components

A
  1. DNA polymerase
  2. Buffer
  3. Nucelotide tri-phosphates
  4. Template DNA
  5. Primers that bind to DNA
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30
Q

Why do we add buffer to PCR?

A

enzymes denature at wrong pH and polymerase needs certain ions to function properly

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31
Q

Where does the template DNA for PCR come from?

A

add bacterial cells to PCR and burst them open

this way they will release their genome into the reaction

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32
Q

Why do we add primers to PCR?

A

DNA polymerase needs to add on to something

We don’t just use RNA polymerase’s primers because we want to control exactly where replication begins

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33
Q

PCR steps

A
  1. Denaturation
  2. Annealing
  3. Extension
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34
Q

Denaturation

A

heat the reaction to ~95 to cause DNA strands to separate

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35
Q

Annealing

A

cool the reaction to ~50 to allow the short DNA primers to base-pair (anneal) to the template

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36
Q

Extension

A

nice middle temperature ~72 where the DNA polymerase we use is active and can copy the template DNA

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37
Q

In 1 round of PCR, how much does the amount of DNA copies we are amplifying increase by?

A

for every 1 round of PCR, the number of DNA doubles

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38
Q

Negative control for PCR

A

no bacterial colony/template

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39
Q

Positive control for PCR

A

straight template DNA that we know works in PCR

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40
Q

What do we add to master mix for PCR?

A

water, buffer, primer 1, primer 2, dNTPs, polymerase

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41
Q

How do we mix small amounts?

A

pipette up and down

do not vortex

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42
Q

How much bacteria do you put into PCR?

A

not too much

too much bacteria will ruin the PCR reaction

just get a little on the tip of your pipette (should not be able to see)

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43
Q

Why do we heat the solution in PCR reactions?

A

causes the bacterial cells to burst and release their DNA

later, denatures DNA

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44
Q

Why do we hold DNA at 72º and then 4º?

A

At 72º, DNA polymerase will copy DNA

At 4º, DNA polymerase will stop copying DNA. DNA is very stable due to lack of reactions

45
Q

How do you cast your gel?

A

Assemble casting tray and comb and ensure water tight seal

Add 3.75 uL SybrGreen to your melted agarose mixture

Swirl to mix

Pour into tray

46
Q

How do you set up gel to load/run?

A

Remove comb

Turn gel so wells are at negative (black) end

Add buffer so completely covers gel

47
Q

Purpose of adding loading dye to samples

A
  1. Makes samples heavier than buffers, so they will sink to the bottom of well
  2. Allows you to visualize migration of the sample through the gel (you are watching DYE move not DNA)
48
Q

How do you know when gel is running?

A

you will see bubbles

49
Q

Why does DNA move to the positively charged end?

A

DNA’s phosphate backbone makes it negatively charged

50
Q

What does agarose do?

A

creates a grid for DNA to travel through

51
Q

Why does smaller DNA go to the positively charged end of gel faster?

A

Longer DNA has a harder time weaving its way through the grid, so it migrates more slowly

52
Q

DNA ladder

A

fragments of DNA of known sizes to compare to our samples

53
Q

SybrGreen

A

fluoresces under UV light when bound to DNA

allows us to visualize the DNA

54
Q

What size DNA do we want to be able to sequence from a PCR?

A

~1500 bp position

55
Q

Candida albican’s ploidy

A

ploidy can change in pathogenic infections or under stress

it is theorized that increasing ploidy, increases the mutation rate and can make candida become pathogenic

56
Q

Candida albican’s research question

A

Does increasing ploidy allow Candida to evolve to host’s environment and become pathogenic?

57
Q

How did we determine mutation rate of Candida?

A

plated Candida with a point mutation for histidine on plates that were negative for histidine

those that grew on HIS- plates were revertants

58
Q

auxotrophs

A

unable to grow on minimal medium

ex: candida that cannot synthesize histidine

59
Q

What do we normalize the revertant rate to?

A

Overall amount of yeast in culture

yeast that grows on YPD plate

60
Q

Why did we need to centrifuge and wash candida?

A

need to get rid of any remaining histidine

61
Q

Mutation rate

A

of revertants / total # of candida

62
Q

of Candida in starting culture

A

= # of colonies * dilution factor * (starting culture volume/volume plated)

63
Q

When is a t-test used?

A

to compare 2 datasets to eachother

64
Q

What is our null-hypothesis?

A

Since there is no expected value our null-hypothesis is just that there is no different between datasets

65
Q

Two numbers needed to perform stats tests

A

number of successes

number of trials (successes + failures)

66
Q

What test do you use to compare results between 2 experiments (groups?)

A

2x2 contingency table

67
Q

What did we analyze in dideoxy sequencing?

A

identified bacteria with and without cellulase activity

68
Q

ddNTPs

A

have no 3’ OH group and cannot further extend

69
Q

Sanger Sequencing Reactions

A

produces thousands of products of all possible lengths

shortest will be primer + one base

longest will be length of template

the last base will always be flourescently labeled with a ddNTP

70
Q

Fluorescent colors of ddNTP

A

ddATP = green

ddGTP = black

ddTTP = red

ddCTP = blue

71
Q

Sanger Sequencing Reactions and gel

A

can run products on a gel to separate them by size

the color of the band tells us what the last base is in each size

can use this to piece together the sequence

72
Q

Why are the ends of Sanger Sequencing chromatogram often messy?

A

difficult to resolve short sequences since they move so quickly

difficult to resolve long sequences since they differ by such a small percentage

73
Q

What types of peaks on a chromotogram do we consider unreliable?

A

multiple peaks for the same base position

no clear end and beginning to peak

peaks not much higher than background levels

74
Q

Basic Local Allignment Search Tool (BLAST)

A

compares your query (from Sanger sequencing) to all sequenced DNA in the NCBI database and searches for matches

75
Q

Identity

A

what percentage of aligned bases matched

76
Q

E-value

A

of sequences aligning this well that you would expect by chance

want a small E-value for more significance

77
Q

Coverage

A

how much of your query sequence aligns with the match

length to length

78
Q

Benefits of Next Generation sequencing

A

Do not need to culture microbes in order to sequence/ID them

Can collect data for thousands of microbes in a single reaction

79
Q

Detriments of Next Generation sequencing

A

complex analyzes

get short reads that can be hard to identify species

you do not have the bacteria to further study

80
Q

Steps of Next Generation sequencing

A
  1. Cluster generation: each cluster came from 1 prepared PCR products
  2. Sequencing: each cluster is sequenced simultaneously
  3. Data analysis: use software to eliminate unreliable data and to identify species
81
Q

What are the two ways to survive a pathogen if you are a C. elegan?

A

Either avoid the pathogen or survive the pathogen

82
Q

Avoidance assay

A

Plate 100 worms on the center of the plate

Count from 20 minutes to 80 minutes the worms on each side of plate

83
Q

Survival assay

A

plate 100 worms directly ontop of Serratia

count how many worms move to e. coli after 24 and 48 hours

84
Q

Survival rate of C. elegans

A

number of worms in e. coli / total plated

85
Q

Technical replicates

A

repeated measurements of the same sample that represent independent measures of random noise associated with protocols or equipment

ex: each student running the avoidance assays in triplicate

helps control for noise in a measurement

86
Q

Biological replicates

A

parallel measurements of biologically distinct samples that capture random biological variation, which may itself be a subject of study or a noise source

ex: each student evolving their own C. elegan strain

helps control for noise in biology

87
Q

What needs to be in a figure caption?

A
  1. title
  2. figure k (if needed)
  3. plot calculations (number of replicates, error bars, data)
  4. statistical information
  5. experimental information (methods)
88
Q

CFU

A

colony forming units

*need to multiply by dilution factor

89
Q

What direction do primers go in?

A

5’ to 3’

90
Q

Why do we need two primers for PCR?

A

need one for each strand

91
Q

TAQ polymerase

A

used in PCR reactions

does not know when to stop, so continues until time runs out/temperature changes

92
Q

Difference between Sanger sequencing and PCR reactions

A

Sanger has only 1 primer and has ddNTPs

93
Q

Does Inverse simpson’s index measure richness or evenness more?

A

evenness

94
Q

species richness

A

number of different species present

95
Q

Which strand does the reverse primer attach to?

A

5’ to 3’ strand

96
Q

What does a larger inverse simpson’s index indicate?

A

greater diversity

97
Q

What does a larger simpson’s index indicate?

A

less diversity

98
Q

When pipetting small volumes, what do you pipette first?

A

large volumes

99
Q

Difference between Sanger sequencing and PCR

A
  1. ddNTP have no 3’ -OH group
  2. ddNTP are flourscently labeled
  3. only use 1 primer in Sanger
100
Q

Replicates in C. elegans project

A

Biological: each student’s passage

Technical: 3 avoidance plates and 3 survival plates with the same sample

101
Q

Plating bacteria for c. elegans project

A

5 uL of Serratia and E. coli on each side were plated with sterile technique

Plates were left agar side down to dry

Incubated for one week under room temperature with agar side up

102
Q

Replicates in Bean beetle project

A

Biological: each group’s unique beetle

Technical: multiple plates

103
Q

Bean beetles technical name

A

Callasobruchus maculatus

104
Q

What are two ways of identifying species?

A

Morphology of bacterial colonies

16s rRNA sequencing

105
Q

What are the two main experimental applications of NGS?

A

whole genome sequencing of a single species

16s sequencing of microbial communities

106
Q

What is a NGS cluster?

A

collection of identical DNA sequences in a flow cell

107
Q

How can NGS be used to calculate species diversity?

A

the proportion of reads for each individual species = the proportion of that species present in a simpson’s index

use the proportion of reads for each individual species to generate an inverse simpson’s index

108
Q

Candida replicates:

A

Biological: different cultures of yeast

Technical: each group’s project from the same stock of yeast

BIO 142 (14 decks)

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