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BIO130 Final > Labs > Flashcards

Labs Flashcards

1
Q

How many microliters and 1 milliliter?

A

1 mL = 1000 microliters

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2
Q

In DNA isolation, why is the material mashed up into small pieces?

A

to provide a greater surface area on which the chemicals can work

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3
Q

What does mechanical disruption do in plant cells?

A

helps break down cell walls but some plants need additional chemical disruptions to extract the DNA

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4
Q

How was the isolation of the nuclei of wheat germs performed?

A
  • Wheat germ was incubated in a mild detergent (NP-40)
  • large debris was filtered out by pouring the sample through cheesecloth
  • the liquid is squeezed out, then centrifuged to separate the nuclei and other insoluble material from soluble or lighter cellular organelles
  • washed the pellet containing the nuclei to remove insoluble materials
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5
Q

How was the basic protocol of separating nuclear DNA from protein and RNA on the wheat germ performed?

A
  • pellet was resuspended and incubated with a strong detergent and incubated at high temps to lyse the nuclear envelope
  • the DNA was separated from the protein in a high salt solution because of their different molecular properties
  • by using a high salt solution and alcohol the DNA will precipitate
  • some contamination with proteins is expected and some RNA also precipitated
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6
Q

How were the cellular proteins denatured and removed from DNA?

A

detergent and heat

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7
Q

When a high salt solution is added, why does DNA separate from RNA?

A

primarily because of the size of DNA, but also because of its double-stranded conformation, and its stability at a more basic pH

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8
Q

How was the refined protocol of separating nuclear DNA from protein and RNA on the wheat germ performed?

A
  • an extra step for protein removal prior to precipitation of DNA occurs
  • the protein precipitation solution is added
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9
Q

What is the protein precipitation solution?

A

a high-salt buffer which affects the solubility of proteins

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10
Q

What is agarose gel electrophoresis used for?

A

to separate molecules via an electric current. The positively charged molecules will move toward negatively charged electrodes, and negatively charged molecules will move toward positively charged electrodes. DNA and RNA are negatively charged and move toward the positively charged electrode

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11
Q

What factor affects the rate of migration for molecules in an electrophoresis?

A
  • shape
  • charge-to-mass ratio
  • size
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12
Q

What is the purpose of SYBR Safe?

A

bind to DNA and enables visualization of DNA bands under UV light

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12
Q

What is the purpose of TBE buffer in the gel electrophoresis?

A

provides ions that carry current and maintain
the pH at a relatively constant value

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13
Q

What are the basic steps of PCR?

A
  • Denature the double-stranded DNA template (high temp)
  • Anneal (base-pairing) of designed DNA primers to sequence to be
    amplified (low temp)
  • Synthesis (extension) of DNA using heat stable DNA polymerase
    (Taq) (med temp)
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14
Q

What does one cycle of PCR result in?

A

the amount of DNA is doubled

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15
Q

What does the PCR buffer do?

A

Maintains pH for Taq polymerase to function, increase yield and efficiency of reaction

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16
Q

What do the primers do during a PCR?

A

Complementary to 3’ end of DNA and anneals to DNA template

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17
Q

What does MgCl2 do during PCR?

A

Provide divalent cations required for activity of DNA polymerases

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18
Q

what is the role of dNTPs during a PCR?

A

Contains dGTP, dCTP, dATP, dTTP nucleotides that can be incorporated into the growing DNA strand

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19
Q

How are genes studied?

A

We sequence genes to convert DNA into letter codes (A, C, T, G)
which we can then study.
* Sequenced genes can then be stored in repositories (e.g.
GenBank) where they can be studied by other scientists.

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20
Q

What are bioinformatic tools used for?

A
  • Compare nucleotide sequences and look for additions,
    deletions, or substitutions.
  • Translate nucleotide sequences into amino acid sequences to
    better understand protein structure and function
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21
Q

How are genes compared to one another?

A

BLAST to search a sequence database (GenBank) for matches to your sequence of interest

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22
Q

How does BLAST access the quality of the match?

A
  • Percent coverage: percent of your sequence that overlaps with a sequence in the database
  • Percent identity: percent of base pairs that are the same between your sequence with a sequence in the database
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23
Q

What is PCR?

A

a method that makes it possible to generate millions of copies of the specific DNA sequence of interest

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24
What is BLASTN used for?
used to compare a nucleotide sequence with a nucleotide database
25
What is BLASTP used for?
used to compare a protein sequence with a database of protein sequences
26
What is Clustal Omega used for?
for carrying out multiple sequence alignment, which generates the alignment between three or more sequences
27
What is a multiple sequence aligment?
the process or the result of sequence alignment of three or more biological sequences, generally protein, DNA, or RNA.
28
What does a Bit Score tell us?
measures the sequence similarity and can be used to compare the alignement scores from different searches
29
What does the E-Value tell us?
helps assess the statistical significance of a given BLAST match (lower E-value= more significant the score) (E-values 0.01 or less are significant
30
What is the P-value?
the probability of obtaining an alignment with the score in question or better
31
How do you calculate the amount of double-stranded DNA that was produced from a PCR?
the initial amount of double-stranded DNA x 2^(# of PCR cycles)
32
What are endonucleases?
enzymes that cleave both strands of the DNA molecule within the DNA polymer
33
What do type 2 restriction enzymes do?
Recognize and bind to short, specific sequences
34
Why are restriction enzymes isolated from bacteria?
it protects them from foreign DNA
35
How many fragments would you get from digesting a plasmid with an enzyme that cuts once?
1, 2 if it was cut twice, 4 if it was cute three times
36
What does molecular cloning require?
- Isolation of DNA sequence (digesting with restrictive enzymes) - Insertion of gene of interest into a vector (producing a recombinant DNA molecule) to propagate
37
What are two naturally occuirng DNA molecules that function as vectors?
- Plasmids: Small circular DNA found in bacteria and yeast. - Able to multiply independently from host DNA. - Can regulate own replication. - Viral nucleic acids: Found in bacteriophages that infect bacteria
38
What is a promoter?
Site where gene expression begins. Any inserted genes are downstream of this promoter
39
what is a multiple cloning site?
A site to insert DNA, the MCS has many restriction enzyme cut sites you can use to cut the plasmid
40
What is the cleave sequence for HindIII?
5- A^AGCTT -3 (^=cut)
41
What is the cleave sequence for BamHI?
5- G^GATCC -3 (^=cut)
42
What is a common feature of restricted enzyme cleavage sites?
the restricted site sequence is the same on each DNA strand when read in the 5' to 3' direction
43
Since bacteria make restriction endonucleases, why do they not digest their own DNA?
bacteria also produce DNA methylase that add one or two methyl groups to bases located within or near the cleavage site
44
What is CRISPR-Cas 9
it's a system tat ises a single guide RNA to guide Cas9 endonuclease to a cleaving site through complementary base pairing. The RNA can be modified to help guid the endonuclease to target and cleave a spot in the genome
45
How can you tell if the correct DNA has been isolated?
- digest the DNA with a cariety of restriction enzymes, then analyze the banding patterns to ensure they match the expected pattern
46
What steps were used to colne the gene in the lab?
- preparation of pure sample of DNA (extracted genomic DNA through PCR) - cleaving of DNA molecules at precise and predicatable location with restriction enzymes - analysis of DNA fragment sizes and sequences - joining of DNA molecules together (ligation) - introduction of DNA into host cell (transformation) - identification of cells that contain recombinant DNA molecules
47
What is Koehler illumination?
an alignment method that allows bright, even illumination across the specimen or field of view and will maximize the resolution of the image
48
What is the objective lense?
Collects light that passes through the specimen, magnifies the resulting image
49
What is the field iris diaphragm?
Controls the amount of light that enters the condenser Direction that light travels
50
What is the condenser?
Focuses light onto the specimen
51
How do you set up a Koehler illumination?
- start with 10x objective - turn on light and adjust light level - adjust distance between ocular lens - focus sample - open field iris diaphrgm all the way - move condenser up close to the slide using condenser focus knob - close the field iris diaphragm until you see it - open the field iris diaphragm and stop as soon as the diaphragm moves out of the fiield of view
52
How do you calculate the maginification?
size of image/ drawing divided by the actual cell
53
What is resolution?
the ability to distinguish between two closely positioned objects
54
What is an ocular reticle?
a tiny ruler etched into the glass lens of one of the microscope's oculars
55
At 10X how many mm are in 100 ocular units? and how many micrometers and mm are in 1 ocular unit
1mm 10 micrometers and 0.01mm
56
At 40X how many mm are in 100 ocular units? and how many mm and micrometers are in 1 ocular unit?
0.25mm 2.5 micrometers and 0.0025 mm
57
If two lines are 100nm apart and the resolution is 200 nm, how would the image appear?
the image will appear as one solid line
58
What is bright-field microscopy used for?
view living cells and microorganisms and fixed tissue (tissue cells are not alive at the end of the fixing process)
59
What is phase-contrast microscopy used for?
induces contrast in wet mounts, unstained and unmounted specimen
60
What is Nomarski immaging used for?
visualize transparent and internal structures
61
what is fluorescence microscopy used for?
labeling/ visualization of discrete part of a cell
62
What is confocal microscopy used for?
determine cellular localization of organelles, cytoskeletal elements and macromolecules; trace specific stationary cells through tissue using 3D reconstruction (cells that move quickly cannot be easily traced)
63
What is transmission electron microscopy used for?
used to view internal structures closely but can also be combined with the metal shadowing process to view external cellular structures
64
What is scanning electron microscopy used for?
view external structures closely. Usually used to view whole cells
65
What is cycloheximide?
an inhibitor of protein synthesis. It binds to eukaryotic ribosome and blocks the elongation phase of translation by inhibiting the action of the EF2 translation eleongation factor
66
What is H0?
a null hypothesis. This is a hypothesis that states a way that there is no difference
67
How can you reject the H0?
if X calculated is greater than X critical H0 can be rejected. Meaning you can conclude with 95% confidence that H1 is true.
68
What is X2 critical?
3.8

BIO130 Final (7 decks)

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