My BIO DAT Prep > Microscopy and Lab Techniques > Flashcards

Microscopy and Lab Techniques Flashcards

(58 cards)

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1
Q

Bacterial cloning

A

Technique that scientists use to clone eukaryotic gene products in prokaryotic cells

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2
Q

Steps of bacterial cloning (5):

A
  1. Processes mRNA for eukaryotic gene of interest is located
  2. Processes mRNA is treated with reverse transcriptase to make cDNA (complementary DNA)
  3. Restriction enzyme and DNA ligase allow cDNA to be incorporated into plasmid (acts as a transfer vector in bacterial cloning)
  4. vector contining gene is taken up by competent bacterial cells
  5. Bacteria that took up vector undergoes transformation (cell genome is changed by addition of DNA from environment)
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3
Q

what is a vector?

A

A piece of DNA that can be taken up by competent cells. This allows the DNA to be replicated, transcribed into mRNA, and translated into protein.

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4
Q

FRAP

A
  • (Fluorescence Recovery After Photobleaching)
  • quantitative measure of how and where biomolecules are moving in a live cell
  • determines whether a membrane protein can freely move around the membrane or if it is a structural component
  • the entire cell is flourescently tagged
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5
Q

Steps of FRAP

A
  1. Scientist measures baseline (normal) fluorescene of sample
  2. An area of a sample is photobleached
  3. Due to cellular dynamics and the moving cytoplasm w/in the cell, photobleaached molecules are replaced by unbleached (fluorescent) molecules overtime
  4. Fluorescence of area is gradually restored and the recovery of fluorescence in a bleached area is measured, informing researchers about movment of membrane proteins
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6
Q

What is the purpose of photobleaching in FRAP?

A

Causes pigmented molecules to permanently lose fluorescence and allows for the quantitative measure of how and where bio molecules move in a live cell

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7
Q

What is the purpse of SDS PAGE?

A

Used to separate proteins by mass

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8
Q

What does SDS do to proteins?

A

SDS (sodium dodecyl sulfate):
* Denatures proteins
* Non-covalent bonds are denatured which causes protein to be linearized (allows protein to be in a non-native, unfolded state)
* Adds a negative charge to the proteins (negative charge is proportional to the proteins mass)
All of which allow for better electrophoresis through polyacrylamide gel

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9
Q

What are the proteins treated with in SDS PAGE?

A

Proteins are treated with primary and secondary antibodies
* primary antibodies will selectively bind to target protein
* secondary antibody will selectively bind to primary antibody (it will also glow a certain color)

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10
Q

Blunt ends

A

Do not have unpaired nucleotides, making them harder to hybridize
They are less common than sticky ends

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11
Q

Transgenic animals

A

Models that researchers use to identify function of a gene

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12
Q

How are transgenic animals used?

A

Gene sequence is taken from one organism and inserted into another organism through recombinant DNA technology

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13
Q

What do transgenic animals allow for?

A

Allow researchers to study the functional purpose of gene sequences
Allow researchers to mass-produce certian medications (ex: people with hemophilia need certain type of clotting factor)

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14
Q

Fluorescence lifetime

A

Refers to the amount of time it takes for an excited molecule to release all its fluorescence

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15
Q

Antibiotic resistance method:

A
  • After restriction enzymes cut DNA, a gene that confers antibiotic resistance is attached to the target gene using ligase
  • Only the cells that have (transformed) picked up and integrated the antibiotic resistance gene/target combo will have antibiotic resistance and will grow on a plate containing an antibiotic
  • Cells that did not pick up this combo will die because they are not resistant to the antibiotic

If cell is still alive, gene of interest is present

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16
Q

Chromotography

A

Separates liquids in a mixture by solubility (differential solubility)

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17
Q

Color change method:

A
  • Vectors containing a gene that make cells blue and restriction enzymes that cut the blue gene will be used
  • If the target gene inserts (transforms) into the blue gene, the blue gene will be inactivated and the cell will appear its normal color (it turns white)
  • If the target gene does not insert into the blue gene, the gene will reattach, remaining active and blue.

White = gene of interest is present

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18
Q

Scanning Electron Microscopy (SEM)

A
  • Captures electrons that are scattered by atoms on the surface of dehydrated samples
  • pro: allows for visualization of high resolution, 3D image of sample surface
  • Con: costly and requires an extensive sample prep (kills sample because they are dried and coated)

SEM = Surface

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19
Q

Purpose of gel electrophoresis

A

To separate DNA, RNA or protein fragments by charge and size in agarose gel

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20
Q

What travels furthest towards the bottom of the gel in gel electrophoresis?

A

Smaller fragments or DNA (or RNA) travel further in the gel because the pores of gel obstruct the movement of larger fragments
Negatively-charged molecules (like DNA) will travel further in the gel because they want to move toward the positive anode at the bottom

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21
Q

Steps of gel electrophoresis

A

1) DNA is cut up into pieces by restriction enzymes
2) DNA is loaded into wells in the agarose gel (at the top of each lane)
* top of gel = negative cathode
* bottom of gel = positive anode
3) Electric field is applied to the gel
4) Negatively charged DNA will be attracted tot he positive end of the gel and smaller fragments will move further from the top of the gel

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22
Q

Pluripotent

A
  • A stem cell can differentiate into any of the three germ layers: endoderm, mesoderm and ectoderm
  • They can give rise to any cell type, but they cannot develop an entire organism because they can’t develop extra embryonic tissue, like the placenta
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23
Q

DNA fingerprinting

A

Identifies individuals through aspects of unique DNA, including RFLPs and STRs (short tandem repeats), group of nucleotides that repeats again and again in a stretch of DNA
Each individual has different RFLPs (except to identical twins)
Used in paternity and forensic cases

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24
Q

Phase contrast microscopes

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A
  • uses light phases and contrast for detailed visualization of thin samples containing live cells
    Cells do not have to fixed, stained, or tagged because these microscopes have good contrast
  • Internal structures may be seen if cell layer is thin enough
  • pro: good resolution and good contrast
  • con: can’t use thick samples and may produce “‘halo effect” around. perimeter of sample
25
How do phase contrast microscopes work?
Light passes through annular ring (creates cones of light that refract once it hits the objects —> refraction is due to differing densities) that creates phase shifts (slight difference in the positioning of light) which creates good contrast and resolution
26
What can occur when using a phase contrast microscope and how it can be overcome?
Halo effect: a large phase shift around the samples edges, distorting the area around the sample The halo effect can be reduced by using a **phase plate** or using **thinner samples**
27
Dark field microscopy
* Allows for visualization of **unstained, live samples** by **increasing contrast** between the sample and the background * high contrast results in the surrounding field appearing very dark
28
How is contrast achieved in dark field microscopy?
By allowing **only** the light that passes through the sample AND scatters to contact light detector - all other light is blocked (including light that is directly transmitted through the sample). **only scattered light** from the sample is transmitted which means that the sample will appear on a completely black back group and therefore the light intensity can be low (con)
29
Stereo microscopes
(Also called dissection microscopes) Offer **low magnification** to observe the **surface** of live samples Whole objects can be viewed in three dimensions
30
Process preventing proteins and structures from degrading in electron microscopy
Fixation
31
Taq polymerase
A special heat-stable DNA polymerase captured from thermophilic bacteria
32
DNA microarray
A chip containing thousands of DNA probes that are complementary to a certain gene sequence
33
What is the purpose of DNA microarray
Allows researchers to determine which genes are expressed (DNA —> mRNA) and which aren’t expressed in a type of cell Fluorescence is emitted when hybridization occurs
34
Steps for DNA microarray
1) isolate specific type of cell from sample and remove all the mRNA (because it represents the active transcription of that cell type) 2) reverse transcriptase synthesizes cDNA from mRNA 3) hybridize cDNA with DNA probes on microarray 4) use analysis machine to examine microarray for fluorescence 5) compare microarray with the sequenced genome
35
Dark field patch stop
Blocks light from the center from entering the object, which creates an outer ring of light in dark field microscopy
36
Condenser lens
Part of the dark field microscope that refocuses the outer ring of light back onto the sample
37
Transformation
A process that occurs when a cells genome is changed by the addition of DNA that was once floating freely in the environment Occurs by bacterial cells picking up naked, free-floating DNA from its environment and incorporating it into its genome
38
Chase phase
Abundance of non-radioactive amino acids are added to the cell This prevents every protein a cell makes from being radioactively labeled, making it easier for researchers to identify the proteins they are interested in studying
39
What is the purpose of polymerase chain reaction (PCR)?
It is a biotechnology process that quickly creates millions of copies of DNA (DNA replication)
40
What are the steps for PCR?
1) **Denaturation**: (~95°C) container is heated which splits DNA *double helix* into separate, *single strands* 2) **Primer annealing**: (~65°C) temperature is lowered which allows *DNA primers* to stick to *single DNA strands* (hydrogen bonding) 3) **Elongation**: (~70°C) nucleotides are added to the *3’ end* of DNA using **Taq polymerase** (Taq uses single DNA strand as template)
41
Genomic library
A collection of the total genomic DNA from a single organism
42
Step in generating a genomic library:
1) **Restriction enzymes** are used to cut the organisms genome into many fragments (plasmids) 2) Fragments are cloned using **PCR** and **DNA ligase** inserts the cloned fragments into bacteria (plasmids preserve the fragments from being broken down) 3) The plasmids are screened for using **antibiotic resistance** or **color changing methods** 4) Once the appropriate plasmids have been isolated from the library, they can be cloned via **bacterial cloning**
43
during FRAP, what is seen when a protein in mobile or immobile?
mobile protein: a high percent recovery and fast mobility immobile protein: low precent recovery and low mobility *percent recovery: the amount of light that returns to the photobleached area relative to the amount present prior to photobleaching
44
reproductive cloning
* produces a genetic copy of an organism * a somatic cell nucleus and an enucleated egg cell from two separate sources are fused together using an *electric pulse* and then grown in culture * **multipotent cell** must be converted to a **totipotent cell**
45
in reproductive cloning, what is the embryo identical to?
somatic cell nucleus of the donor
46
antibiotic resistance screening
used in order to test if bacteria successfully took in the plasmid- plasmids carry genes that confer antibiotic resistance to bacteria
47
to perform gel electrophoresis on proteins, what must be added?
SDS (a chemical that denatures proteins and gives them all a uniform negative charge) must be added because proteins R groups causes them to have different charges that will affect their migration patterns
48
what can be viewed with a light microscope?
mitochondria, bacteria, red blood cell, animal cell, plant cell, pollen, human egg, frog egg, fruit fly *generally no viruses because they are too small*
49
what can be viewed with an electron microscope?
atoms, molecules, proteins, lipids, viruses
50
stereomicroscope
* also called light microscopes or dissection microscopes * uses visible light to view the surface of an object - creates a 3D image of the samples surface * has low magnification and low resolution
51
compound microscope
* has multiple lenses * uses visible light to create 2D images of a single cell layer with its internal structures * can view live cells, but has poor contrast without fixing and staining so usually requires staining
52
what was the purpose of the Avery-MacLeod-McCarty experiment?
it was conducted to discover what was the cause of bacterial transformation in Griffith's experiment
53
what did the Avery-MacLeod-McCarty experiment reveal?
it revealed that DNA is heritable in bacterial transformation
54
transmission electron microscopy (TEM)
* visulaizes **high resolution, 2D** image of samples **internal surface** * captures electrons that are transmitted through a **thin slice** of a sample * pro: can view internal structures with high detail * con: costly and extensive sample prep (kills sample) | **T**EM = **T**wo-D
55
cryo-SEM
* sample is frozen in **liquid nitrogen** * freezing process provides 3D image of sample surface in its **more natural form** * pro: high resolution * con: extensive prep kills sample, freezing can cause artifacts
56
electron tomography
* not a type of microscope, but an analytical lab technique that stitches together 2D photos to create 3D image of samples internal sturctures * 2D images are obtained using **TEM** (sandwhiches TEM images together) * pro: can look at objects in 3D and see objects relative to one another * con: extensive prep (kills sample) and costly
57
what are the benifits of cDNA lacking introns?
* cDNA is only composed of exons and no introns, so splicing is not required * enables effective transcription and translation of genes * extremely useful for bacterial cloning, expressing eukaryotic genes in prokaryotic cells like bacteria which lack splicing activity
58
in vitro mutagenesis
* procedure in which mutations are introduced into a **cloned gene** which will result in **phenotypic changes** * these changes can be used to determine the function of the gene of interest * ex: knockout mice: gene of interest was knocked out and phenotypic changes were observed
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