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1
Q

Define Solution

A

A mixture of a compound (solute) in a liquid (solvent). The amount (mass) of the compound in the total volume of solution is the concentration of the solute. A solution can have more than one compound and each compound has a concentration.

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2
Q

What is the equation for concentration and what are some common units?

A

Concentration= Mass/Total Volume; Mass in Grams/Volume in Liters

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3
Q

What is th equation for molarity?

A

Molarity=Moles of solute/liters of solution

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4
Q

How is concentration expressed in this course?

A

X. A 1X solution is ready for use, while a 10X stock solution needs to be diluted 10 fold.

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5
Q

What is the difference between a Stock Solution and a working solution?

A

Stock Solutions are anything greater than 1X; working solutions exclusively have a 1X concentration.

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6
Q

What is the formula used to calculate a dilution?

A

C1V1=C2V2
C1= stock concentration; V1=stock volume
C2=final concentrations ; V2= final volume

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7
Q

What are the ranges for the micropipettes?

A

P2: 0.5-2
P20: 2-20
P200: 20-2000
P1000: 200-1000

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8
Q

How do you calculate the volume of Water in a problem involving C1V1=C2V2?

A

Once the V1 is determined through using the formula, you subtract the V1 from the V2 value to find the volume of water.

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9
Q

How do you read the display of a P2?

A

You divide the entire thing by 100.

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10
Q

How do you read the display of a P20?

A

You divide the entire thing by 10.

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11
Q

How do you read the display of a P200?

A

You read it as it is; no multiplication or division is required.

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12
Q

How do you read the display of a P1000?

A

You multiply the entire thing by 10.

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13
Q

Define Genetic Information Transfer (GIT)

A

The process required to produce a protein or functional RNA using the information stored in genes.

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14
Q

How is the process of GIT started?

A

The process begins when a gene is turned on or expressed. The DNA is then transcribed into pre-mRNA using the enzyme RNA polymerase and the process of transcription.

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15
Q

How is pre-mRNA modified?

A

A guanine cap is added to the 5’ end and a poly A tail is added to the 3’ end.

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16
Q

Define Introns

A

A segment of DNA or RNA molecule which deos not code for proteins.

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17
Q

What are the coding and non-coding regions called respectively?

A

Non-Coding: Introns
Coding: Exons

NICE

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18
Q

What happens to introns and exons in the splicing process?

A

Introns are removed and exons are joined together.

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19
Q

Where does splicing occur?

A

In the spliceosome, a structure consisted of proteins and RNA.

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20
Q

What happens in the process of translation?

A

In the cytoplasm, mRNA meets up with the machine that will decode it and produce a string of amino acids. This machine is the ribosome and contains both proteins and rRNAs. The sequence of the mRNA is read in groups of three nucleotides (a codon) and the tRNA with the complementary sequence enters the ribosome. An amino acid is attached to the tRNA and the ribosomes transfer the amino acid to the growing protein. When the ribosome reaches a stop codon (UAG,UGA, UAA), the protein is released to perform its function and the mRNA can be transplanted again.

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21
Q

Why are control reactions important?

A

If the experiment does not work, the controls provide reasons why.

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22
Q

What are the two types of controls?

A

Positive-Negative

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23
Q

What should be seen with negative controls?

A

No result should be observed. If anything is observed in the negative control, it suggests that an error occurred during the experimental set-up or the reagents used were contaminated.

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24
Q

How are positive controls used?

A

They are used to confirm positively characterized results in a new sample or to confirm that all reagents were added properly.

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25
What do all PCR reactions contain?
Enzymes- dNTPs- Water- Buffer. EDB-W
26
What are the variable reagents in the first experiment?
Primers
27
What is the function of primers?
The primers direct the polymerase to the region of DNA for copying. In the first experiment, the primers are intended to bind to the Actin Gene.
28
Define Transcription
The process by which a cell makes an RNA copy of a piece of DNA. This RNA copy, called mRNA, carries the genetic information needed to make proteins in a cell. It carries the information from the DNA in the nucleus of the cell to the cytoplasm, where proteins are made.
29
Define Translation
The process by which a cell makes proteins using the genetic information carried in the mRNA. The mRNA is made by copying DNA, and the information it carries tells the cell how to link the amino acids together to from proteins.
30
Define DNA
The molecules inside cells that carry genetic information and pass it from one generation to the next.
31
Define pre-mRNA
The first transcript from a protein coding gene is called pre-mRNA and contains both introns and exons. Pre-mRNA requires splicing (removal) of the introns to produce the final mRNA molecule containing only exons.
32
Define tRNA
A small molecule that plays a key role in the synthesis of proteins. It serves as a link between the mRNA and the growing chain of amino acids that make up a protein. Each time an amino acid is added to the chain, a specific tRNA pairs with its complementary sequence on the mRNA molecule, ensuring that the appropriate amino acid is inserted into the protein being synthesized.
33
Define Ribosome
An intercellular structure made up of both RNA and proteins which is the site of protein synthesis in a cell.
34
Define DNA Polymerase
A type on enzyme that is responsible for forming new copies of DNA, in the form of nucleic acids.
35
Define RNA Polymerase
An enzyme that is responsible for copying a DNA sequence into an RNA sequence, during the process of transcription.
36
In the most basic terms, what are the processes of Transcription and Translation? Which comes first?
Transcription comes first, then translation. Transcription is the process of DNA turning into mRNA. Translation is the process of mRNA turning into protein.
37
In which site of the cell are proteins manufactured?
Ribosomes
38
Define Enzymes
Proteins that act as biological catalysts by accelerating chemical reactions.
39
How is a gene defined in this class?
A region of DNA that codes for proteins.
40
What are genes consisted of?
Introns and Exons
41
What is removed during the splicing process?
Introns
42
What is going to be compared in the first experiment?
The cDNA and the gDNA of the Actin Gene
43
What technique is going to be relied on in order to determine the gene structure?
PCR
44
Can PCR work with a single stranded piece of DNA?
No
45
What are the 3 traits of DNA?
Variable-Dynamic-Heritable. VDH
46
One of the major changes in DNA are _____
Mutations
47
Define Vertical Transmission
The passing down of traits from one generation to the next.
48
What are the spaces in between exons called?
Introns
49
_____ control the expression of a gene.
Promoters
50
cDNA is a copy of _____.
mRNA
51
What is the relationship between the % associated with the gel and the density of the gel?
The higher the %, the more dense the gel; the lower the %, the better it is to discriminate larger bonds.
52
What aspect of DNA gives it a certain charge? What properties does it give it?
Sugar phosphate backbone of the DNA gives it a negative charge; it gives it acidic properties.
53
How does the increased presence of G’s and C’s in a DNA sequence impact the annealing and the denaturing process?
The more G’s and C’s are present in a sequence, the lower the temperature required for annexing and the higher the temperature required for denaturation.
54
What is the temperature range for annealing?
50s to 60s.
55
What is the temperature range for Denaturation?
95 or 98
56
What is the temperature for Extension?
72
57
Define Denaturation in PCR
When the double-stranded DNA template is heated to separate it into 2 single strands.
58
Define Annealing in PCR.
When the temperature is lowered to enable the DNA primers to attach to the template DNA.
59
Define Extension in PCR.
When the temperature is raised and the new strand of DNA is made by the Taq Polymerase enzyme.
60
Which DNA of the Actin Gene should be longer?
GDNA due to the presence of introns and exons.
61
When doing alignment in the nCBI blast, how should you sort it?
Sort by Query Start
62
In gene diagrams, what are the numbers on top?
Query # Ranges
63
When you get one band as a result of the experiment, what happened?
You forgot to add enough DNA, you lost the sample, there was improper loading, or there was leakage.
64
If there are no bands at all in the first experiment, what happened?
You forgot to add the primers.
65
When doing alignment, what should be done in terms of placement of sequences?
The longer sequence should always be on top.
66
Define Primer
A short single stranded RNA used by all living organisms in the initiation of DNA synthesis.
67
Define Polymorphism
If two individuals have different sequences at the same place in the genome, it is called a polymorphism.
68
What is a difference on one nucleotide in a polymorphism called?
Single Nucleotide Polymorphism (SNP)
69
What is an example of an SNP in humans? Go in detail.
The mutation that results in sickle cell anemia is due to SNP: the normal allele has a glutamic acid codon (GAG) whereas the mutant allele has a valine (GTG) codon at the exact same place in the protein. This is an example of a non-synonymous mutation and, in this case, it is detrimental to protein function.
70
Define Non-Synonymous Mutation.
A nucleotide mutation that alters the amino acid sequence of a protein.
71
Define Synonymous Mutation
A nucleotide mutation that does not alter the amino acid acid sequence of a protein.
72
What are the different types of polymorphisms?
Single Nucleotide Polymorphisms (SNP)- Insertion/Deletion (Indels)-Transposable Elements (TE)
73
Define Insertion/Deletion
Occurs when one or more nucleotide of DNA is present in one individual and absent in another.
74
SNP’s and Indels are the results of errors during the _____ process.
DNA Replication
75
Define Transposable Elements
DNA sequences that move from one location in the genome to another. They are divided into autonomous and non-autonomous.
76
Explain everything about Autonomous and Non-Autonomous Transposable Elements
The autonomous element contains the gene encoding transposase (TPase) enzyme necessary for the movement of both the autonomous and non autonomous elements. The non-autonomous elements lacks a functional TPase and depends on the autonomous element for movement.
77
What do the arrows at the end of the autonomous and non-autonomous transposable elements represent?
Terminal Inverted Repeats
78
Define Terminal Inverted Repeats (TIRs)
A single stranded sequence of nucleotides followed downstream by its reverse complement. TIRs are the binding sites of TPase and it is the action of the transposase enzyme that results in insertion polymorphism. When a TE moves, polymorphism is generated at the site that it left and at the new insertion site.
79
One of the big surprises of the genomic era is that TEs account for over _____ of genomic DNA of many species. Most TEs in a genome are _____ meaning that they can no longer ______. Active TEs have often evolved mechanisms to avoid harming the organism. They insert between genes, into other TEs, or into introns.
50%-Inactive-Move
80
In the second experiment, B73 is used as a _____.
Positive Control
81
What does diploid mean in the second experiment?
That the 169020 locus occurs twice in the genome.
82
The strains without TEs are going to be _____ in length.
Shorter
83
What does the presence of 2 bands entail?
That there is a +/- insertion of a transposable element; heterozygous.
84
What is the function of primers in PCR?
The primers direct the polymerase to the region of DNA for copying.
85
What is the function of Taq Polymerase in PCR?
The function of Taq Polymerase is to amplify the DNA for the production of multiple copies of DNA.
86
How is cDNA made?
It is synthesized from mRNA.
87
Define Reverse Transcriptase
A DNA polymerase that uses RNA instead of DNA as a template.
88
Define Transcription Factors
Proteins that control genes; they bind to DNA near the beginning of a gene.
89
Define Promoters
Promoters are a DNA sequence; it’s where the RNA polymerase binds to; they regulate the functionality and the expression of genes.
90
When a transcription factor is present in a gene, what happens?
It prevents the binding of RNA Polymerase. If a small molecule (like a sugar or a hormone) binds to the transcription factor, the TF comes off of the gene and allows RNA polymerase to bind to the promoter to begin transcription.
91
Define Constitutive Promoters
Promoters that are always on.
92
Define Conditional Promoters
Promoters that are activated or turned on as long as certain conditions are met; it could be the presence of a hormone or a sugar.
93
What is GFP?
Green Fluorescent Protein; it is the conditional promoter in the third experiment.
94
Why is GFP important for certain scientific studies?
Because it allows for genes to be visualized without damaging the tissue.
95
What are the sugars included in this lab?
Arabinose-Lactose-Galactose
96
Define Transformationq
When any DNA is taken from one organism and placed into another organism.
97
Define Plasmid
Small circular DNA. `
98
Define Transformants
The cells that only take up a plasmid.
99
What are the two genes present in the pGlo plasmid?
Ampicillin Resistance (AMP) and GFP The promoter for AMP is constitutive and the promoter for GFP is conditional.
100
What does it mean for a cell to be competent?
The ability of a cell to take up extra cellular DNA from its environment.
101
How are the plasmids in the 3rd experiment made competent?
The are mixed with bacterial cells treated with calcium chloride.
102
What is the reason that bacteria are alive?
Ampicillin Resistance
103
For the bacteria to be able to grow, which gene needs to be expressed?
Ampicillin Resistance
104
What are the characteristics of UV light?
Low wavelength/ High Energy
105
What are characteristics of GFP light?
High Wavelength/ Low Energy
106
What is the sugar needed to turn on the GFP?
Arabinose
107
What is the portion present in plates that is necessary for bacterial growth?
LB Broth
108
What does it mean for a gene to be a reporter gene?
It tells us where and when the gene is expressed.
109
What is ampicillin?
An antibiotic that kills bacteria.
110
The process of gene expression is regulated by _____.
Promoters
111
Define Chromosome
A combination of DNA and its associated proteins.
112
Define Sex Chromosomes
Chromosomes that are involved in sex determination and segregate differently between the sexes. Many mammals have an XY sex determination where the XX is the female and the XY is the male.
113
Define the SRY gene
The gene on the Y chromosome that acts as a mater switch when it comes to turning on male development and suppressing female development.
114
What are some important things to remember about mammalian Y chromosomes.
They have been evolving in males ONLY for millions of years. Have lost most of the genes once shared with the X chromosome. Have retained a core set of ancestral genes, some of which are found in all placental mammals.
115
What are the chromosomes located in the male and female creeping voles?
XX in male voles; X in female voles.
116
What were some additional discoveries about the creeping voles?
The second sex chromosome in males has all the same genes a the large X that’s in both sexes. Both X chromosomes have all or most of the ancestral Y genes fused to one end. Female creeping voles have 1 copy of all the X chromosomes genes in addition to all the genes that have survived fro over a 100 million years on Y chromosomes.
117
Holistically speaking, what was done in this lab?
Initially, RNA was extracted from different vole tissues through using an RNeasy kit. After that was done, the quality and the quantity of the RNA was tested through gel electrophoresis. If the bands were distinct and clear, that means the RNA was of high quality. If the bands were smeared and it clear, it means the RNA was of low quality. From there, cDNA was made through using iScript for the process of Reverse Transcription. Different dilutions of the cDNA were made; specifically, 1/10 cDNA, 1/100 cDNA, stock cDNA, and water were used. The first PCR was an Actin PCR; it was conducted with the intent of determining which dilution of cDNA was the best to conduct primer testing done. Finally, after that was determined, everyone was assigned different primers and tested them on the same tissues. The appearance of bands means that the gene was expressed in the respective tissue and no bands appearing meant that the gene was not expressed in that particular tissue.

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