Lab Final Flashcards
What must be included in a lab notebook?
Title Date Purpose Methods Data Conclusions
C. elegan control
heat killed S. Marcescens
cannot infect C. elegans, but smells good to them
C. elegans experimental plate
Serratia on one side
E. coli on the other
What should be labeled on a plate for c. elegans?
Date Group name Type of plate (evolution or control) Lab section Lab room number Passage number
Parts of a scientific abstract
Introduction
Materials and Methods
Findings/results
Discussion
Components of the introduction of a scientific abstract
Context, knowledge gap, and objectives
Where does the microbiome come from?
Fetus is probably sterile
Bacteria is acquired at birth, changes as the human develops/changes
Bacterial composition is highly variable
What is the microbiome thought to be determined by?
Diet
Genetics/immune system
environment
How can the microbiome negatively affect a person’s health?
certain species have been identified in gastrointensional distress, weight gain, and neurological diseases
Is most bacteria in the microbiome beneficial?
yes
helps with food digestion, wound healing, gut barrier function
Why is the bean beetle a good model organism?
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
Broad and specific research questions of bean beetle project
Broad: How does diet affect the microbiome?
General: Do beetles raised on different bean types have different microbiomes?
PEA plate
selectively cultivates Gram positive microbes
P = Positive
EMB plate
selectively cultivates Gram negative microbes
Cellulose plates
contains indicator that will show color change around microbes that can digest cellulose
Gram negative bacteria
have an outer membrane ontop of their peptidoglycan cell wall
Why do we sterilize the bean beetles?
sterilize their outside, so we only culture internal bacteria
Why do we plate both diluted and undiluted beetle microbiomes?
Some beetles have tons of microbes while others have much less
What should be labeled on the bean beetle plates?
Group name date lab section lab room beetle type beetle # dilution type of plate
What are the 2 ways that scientists think about diversity?
- Species richness
2. Species evenness
Species richness
total number of species present
Species evenness
the numbers of each species are pretty even
Inverse Simpson’s Index
1/ (# of species A /total)^2 + (# of species B/total)^2…
What plates do we use for diversity measures?
EMB and PEA plates
since they show gram-negative vs. gram-positive bacteria
Morphological data on bacterial colonies
- Color
- Gloss
- Colony size
- Shape
- Elevation
How to determine what part of genome to sequence?
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
Why is the 16s rRNA gene commonly sequenced in bacterial DNA?
the 16s gene is conserved across all bacterial species and has conserved and variable regions
How do we amplify DNA for sequencing?
Polymerase Chain Reaction (PCR)
PCR components
- DNA polymerase
- Buffer
- Nucelotide tri-phosphates
- Template DNA
- Primers that bind to DNA
Why do we add buffer to PCR?
enzymes denature at wrong pH and polymerase needs certain ions to function properly
Where does the template DNA for PCR come from?
add bacterial cells to PCR and burst them open
this way they will release their genome into the reaction
Why do we add primers to PCR?
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
PCR steps
- Denaturation
- Annealing
- Extension
Denaturation
heat the reaction to ~95 to cause DNA strands to separate
Annealing
cool the reaction to ~50 to allow the short DNA primers to base-pair (anneal) to the template
Extension
nice middle temperature ~72 where the DNA polymerase we use is active and can copy the template DNA
In 1 round of PCR, how much does the amount of DNA copies we are amplifying increase by?
for every 1 round of PCR, the number of DNA doubles
Negative control for PCR
no bacterial colony/template
Positive control for PCR
straight template DNA that we know works in PCR
What do we add to master mix for PCR?
water, buffer, primer 1, primer 2, dNTPs, polymerase
How do we mix small amounts?
pipette up and down
do not vortex
How much bacteria do you put into PCR?
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)
Why do we heat the solution in PCR reactions?
causes the bacterial cells to burst and release their DNA
later, denatures DNA
Why do we hold DNA at 72º and then 4º?
At 72º, DNA polymerase will copy DNA
At 4º, DNA polymerase will stop copying DNA. DNA is very stable due to lack of reactions
How do you cast your gel?
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
How do you set up gel to load/run?
Remove comb
Turn gel so wells are at negative (black) end
Add buffer so completely covers gel
Purpose of adding loading dye to samples
- Makes samples heavier than buffers, so they will sink to the bottom of well
- Allows you to visualize migration of the sample through the gel (you are watching DYE move not DNA)
How do you know when gel is running?
you will see bubbles
Why does DNA move to the positively charged end?
DNA’s phosphate backbone makes it negatively charged
What does agarose do?
creates a grid for DNA to travel through
Why does smaller DNA go to the positively charged end of gel faster?
Longer DNA has a harder time weaving its way through the grid, so it migrates more slowly
DNA ladder
fragments of DNA of known sizes to compare to our samples
SybrGreen
fluoresces under UV light when bound to DNA
allows us to visualize the DNA
What size DNA do we want to be able to sequence from a PCR?
~1500 bp position
Candida albican’s ploidy
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
Candida albican’s research question
Does increasing ploidy allow Candida to evolve to host’s environment and become pathogenic?
How did we determine mutation rate of Candida?
plated Candida with a point mutation for histidine on plates that were negative for histidine
those that grew on HIS- plates were revertants
auxotrophs
unable to grow on minimal medium
ex: candida that cannot synthesize histidine
What do we normalize the revertant rate to?
Overall amount of yeast in culture
yeast that grows on YPD plate
Why did we need to centrifuge and wash candida?
need to get rid of any remaining histidine
Mutation rate
of revertants / total # of candida
of Candida in starting culture
= # of colonies * dilution factor * (starting culture volume/volume plated)
When is a t-test used?
to compare 2 datasets to eachother
What is our null-hypothesis?
Since there is no expected value our null-hypothesis is just that there is no different between datasets
Two numbers needed to perform stats tests
number of successes
number of trials (successes + failures)
What test do you use to compare results between 2 experiments (groups?)
2x2 contingency table
What did we analyze in dideoxy sequencing?
identified bacteria with and without cellulase activity
ddNTPs
have no 3’ OH group and cannot further extend
Sanger Sequencing Reactions
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
Fluorescent colors of ddNTP
ddATP = green
ddGTP = black
ddTTP = red
ddCTP = blue
Sanger Sequencing Reactions and gel
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
Why are the ends of Sanger Sequencing chromatogram often messy?
difficult to resolve short sequences since they move so quickly
difficult to resolve long sequences since they differ by such a small percentage
What types of peaks on a chromotogram do we consider unreliable?
multiple peaks for the same base position
no clear end and beginning to peak
peaks not much higher than background levels
Basic Local Allignment Search Tool (BLAST)
compares your query (from Sanger sequencing) to all sequenced DNA in the NCBI database and searches for matches
Identity
what percentage of aligned bases matched
E-value
of sequences aligning this well that you would expect by chance
want a small E-value for more significance
Coverage
how much of your query sequence aligns with the match
length to length
Benefits of Next Generation sequencing
Do not need to culture microbes in order to sequence/ID them
Can collect data for thousands of microbes in a single reaction
Detriments of Next Generation sequencing
complex analyzes
get short reads that can be hard to identify species
you do not have the bacteria to further study
Steps of Next Generation sequencing
- Cluster generation: each cluster came from 1 prepared PCR products
- Sequencing: each cluster is sequenced simultaneously
- Data analysis: use software to eliminate unreliable data and to identify species
What are the two ways to survive a pathogen if you are a C. elegan?
Either avoid the pathogen or survive the pathogen
Avoidance assay
Plate 100 worms on the center of the plate
Count from 20 minutes to 80 minutes the worms on each side of plate
Survival assay
plate 100 worms directly ontop of Serratia
count how many worms move to e. coli after 24 and 48 hours
Survival rate of C. elegans
number of worms in e. coli / total plated
Technical replicates
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
Biological replicates
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
What needs to be in a figure caption?
- title
- figure k (if needed)
- plot calculations (number of replicates, error bars, data)
- statistical information
- experimental information (methods)
CFU
colony forming units
*need to multiply by dilution factor
What direction do primers go in?
5’ to 3’
Why do we need two primers for PCR?
need one for each strand
TAQ polymerase
used in PCR reactions
does not know when to stop, so continues until time runs out/temperature changes
Difference between Sanger sequencing and PCR reactions
Sanger has only 1 primer and has ddNTPs
Does Inverse simpson’s index measure richness or evenness more?
evenness
species richness
number of different species present
Which strand does the reverse primer attach to?
5’ to 3’ strand
What does a larger inverse simpson’s index indicate?
greater diversity
What does a larger simpson’s index indicate?
less diversity
When pipetting small volumes, what do you pipette first?
large volumes
Difference between Sanger sequencing and PCR
- ddNTP have no 3’ -OH group
- ddNTP are flourscently labeled
- only use 1 primer in Sanger
Replicates in C. elegans project
Biological: each student’s passage
Technical: 3 avoidance plates and 3 survival plates with the same sample
Plating bacteria for c. elegans project
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
Replicates in Bean beetle project
Biological: each group’s unique beetle
Technical: multiple plates
Bean beetles technical name
Callasobruchus maculatus
What are two ways of identifying species?
Morphology of bacterial colonies
16s rRNA sequencing
What are the two main experimental applications of NGS?
whole genome sequencing of a single species
16s sequencing of microbial communities
What is a NGS cluster?
collection of identical DNA sequences in a flow cell
How can NGS be used to calculate species diversity?
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
Candida replicates:
Biological: different cultures of yeast
Technical: each group’s project from the same stock of yeast
