Bio 150 Lab Final Exam Flashcards
main parts of the flow chart
exploration, testing ideas, community analysis and feedback, benefits and outcomes
community analysis and feedback examples
replication, publication, discussing with colleagues, theory building
testing ideas examples
gather and interpret data
benefits and outcomes examples
develop technology, inform policy, and build knowledge
exploration and discovery examples
explore the literature, ask questions, make observations, share data and ideas, find inspiration
what is the entryway into the process of science?
exploration and discovery
what part of the flow chart did we do on the first day of class?
exploration and discovery
scholarly literature
disseminate information, peer-reviewed, reliable but not always accessible
non scholarly literature
want to make money, not as reliable, not peer reviews, more accessible
primary literature
original research from original people, usually peer reviewed, best for citing, has a methods section (example: journal articles)
secondary literature
second hand summary, sometimes peer reviewed, lacks method section, ok for citing. (example: scientific books)
tertiary literature
third hand’ general background information, not written by experts and rarely peer reviewed, should be avoided. (example: tertiary literature, encyclopedia and textbooks)
what does CRAAP stand for
currency
relevance
accuracy
authority
purpose
scientific testable questions
addressed the issues/ questions using data
non scientific testable questions
addresses issues/ questions using values, beliefs or judgements
independent variable
variation does not depend on another variable
dependent variable
does depend on variation of independent variable
control
designed to show change
descriptive study
scientist is not testing a hypothesis; they are simply making observations
non experimental study
variable is not manipulated and just gathering data to test a hypothesis, examine if there is a relationship between the two variables but not a casual relationship, hypothesis is tested
experimental study
hypothesis tested, variable is manipulated to measure response of dependent variable, good at establishing casual relationships
what studies require independent and dependent variables?
experimental and non experimental studies
correlation
a non experimental study that does not determine causation
pipettor parts
plunger, tip ejector, shaft, pipette tip
how to use a pipette
vertical position
correct volume is middle to top volume range
3 basic steps of PCR
denature, anneal, extend
anneal
temperature reduced to 55 and the PCR primer anneal/ base pair with the DNA template
denature
heat is increase to 94 and the hydrogen bonds break so the two strands separate
extend
heated up to 72. TAQ polymerase binds to the primers and new DNA begins to be synthesized
major ingredients in PCR reaction
DNA template
PCR primers
DNA polymerase
Deoxyribonucelotide (dNTPs)
dna template role in PCR reaction
dna is copied
PCR primers role in PCR reaction
PCRs will anneal to the template DNA and that will be the starting point for DNA replication. They also select the region of DNA that will be amplified
what primers are needed for PCR reaction
forward and reverse
DNA polymerase role in PCR reaction
an enzyme that can synthesize new DNA. TAQ polymerase is used because it can withstand the heat and allows us to perform repeated rounds of DNA replication
dNTPs
monomers/ building blocks of DNA. DNA polymerase links dNTPs together into a new DNA strand
how is exponential growth related to PCR
the theoretical number of copies of DNA produced in a PCR reaction from a single template can be calculated as 2 to the # of pcr cycles
why are positive and negative controls needed?
the positive control will tell you if DNA was amplified and demonstrates all reaction components are present and working
the negative control will tell you if there are contaminants
what goes into the master mix
- 15 ul x the number of samples + 1 extra sample
- dna extract DOES NOT go into the master mix
- 28 ul of mix will go into individual PCR reaction tubes before any DNA is added
DNA purity
other substances in DNA extracts that are not DNA (ie: salts and proteins)
PCR inhibitor
molecules that interfere with DNA amplification in PCR
DNA concentration
can not be to high or to low otherwise the PCR reaction will fail
factors that reduce DNA purity
dna extract solution carried over from extraction process like ethanol, proteins and lipids
how DNA concentration influences the likelihood of PCR success
not enough can cause the reaction to fail because enough copies might not be produced
to much might cause amplification to stop before exponential phase of the reaction is reached
how DNA purity influences the likelihood of PCR success
to many other substances may be read
how does spectrophotometry work
it increases the amount and wavelength of light absorbed/ transmitted through a sample; it will give the concentration of different compounds in a solution
A260:A280 ratio
1.8
A260:A230 ratio
2-2.2
ideal DNA concentration for PCR
50-250 ng/ul
wavelength DNA is absorbed at
260 nm
is the A260:A280 ratio is low and DNA concentration is high what should happen to the sample?
it should be diluted
if the ratios are too low what should happen to the sample?
it should be kept but a new DNA extraction should be done
wavelength proteins are absorbed at
280 nm
wavelength salts are absorbed at
230 nm
wavelength carbs are absorbed at
230 nm
what does A260/A280 measure
proteins
what does A260/A230 measure
sugars and carbs
equation to dilute a sample of known DNA concentration
(starting concentration)(starting volume)= (final concentration)(final volume)
main goals of gel eletrophoresis
to see if a PCR produced a product and see if the product size is the expected size
why we use a marker (ladder)
the size of PCR products can be estimated in other lanes
why we use GelRed stain
DNA intercalator and flourences under UV light
why we use positive controls
to know the size
why we use negative controls
to see if there are contaminations
typical size (bp) of a DNA barcode sequence
.5-.8 kp
primer dimers
they form when annealing occurs between primers
how can you tell primer dimers
the bottom of the gel is very blurry
gDNA (definition)
too much template added to the reaction, low molecular weight, fragmented DNA may anneal and self prime increasing concentration
gDNA (spotting it)
long smears that are vertical
how to increase gDNA
dilute the template and repeat PCR
major steps of DNA barcoding
specimen, collection data, tissue sample, photos, run a gel, DNA subway
steps of running a gel
extract DNA, PCR amplification, sequence
parts of DNA subway
sequence viewer, sequence trimmer, pair builder, consensus builder, blastin, muscle, phylip nj, phylip ml
primer sets for plants, inverterbae/ animals, frogs/ bacteria, fungi
plants: Rbcl
inverterbae/ animals: COI
frogs/ bacteria: 16s
fungi: ITS
single read
before the sequence is trimmed
trimmed read
after the sequence has been trimmed
consensus sequence
combination of trimmed and reverse DNA sequences that is typically more accurate than each sequence alone; allows you to make a more accurate DNA barcoding sequence
bit score
summary statistic based on the length of the alignment and number of mismatches
aln. length
length of sequence used to make match
e score
likelihood that the match BLAST made between our query sequence and your match could happen by chance
mismatches
number of non common bases between query and bit score
