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Genetics Lab 2
Terms in this set (146)
The first isolation of DNA was done in 1869 by
Isolation and purification of DNA can be described as:
from in vivo to in vitro
DNA isolation can be divided into 3 stages
-Cell disruption/ lysis
-Removal of proteins, polysaccharides, lipids and other contaminants
-Recovery of DNA
____ ____ ____ ____, commonly referred to as cell disruption or cell lysis, to expose the DNA within.
Breaking the cells open
Cell lysis is commonly achieved by chemical and physical methods:____, ____, or ____ the sample.
blending, grinding or sonicating
Removing membrane lipids by adding a ____ or ____ which also serves in cell lysis.
Example of detergent:
Sodium Dodecyl Sulfate
Removal of Proteins: use ____-____ ____
Phenol-chloroform extraction: the ____ denatures the proteins
Proteins and organic contaminants remain in the ___ phase (bottom fraction) while nucleic acids are contained in the____ phase (top fraction)
DNA purification from detergents, proteins, salts and reagents used during cell lysis step. The most commonly used procedures are:
a. ____ ____ usually by ice-cold ethanol or isopropanol. Since DNA is ____ in these alcohols, it will aggregate together, giving a pellet upon centrifugation. Precipitation of DNA is improved by increasing of ____ strength, usually by adding ____ ____
Ethanol precipitation; insoluble
ionic; sodium acetate.
The most commonly used procedures are:
b. _____-____ extraction
The most commonly used procedures are:
c. _____ purification that relies on the fact that the nucleic acid may bind (_____) to the solid phase (____ or other) depending on the pH and the salt content of the buffer
Refinements of the technique include adding a ______ ______ to sequester divalent cations such as, Mg2+ and Ca2+, which prevents enzymes like DNase from degrading the DNA.
-Storage of ____ ____ under buffered conditions.
-Composed of ____ and ____
____ is a chelating agent that inhibits the activity of DNAses
Tris-HCl and EDTA.
CHAPTER 4: PLASMID DNA
A ____ is an extrachromosomal double-stranded DNA molecule that replicates independently from the chromosomes within bacterial cells
Plasmids used for DNA cloning usually have been engineered to contain:
-a number of convenient ___ ___
-a ____ ____ to select for its presence in the host cell
Alkaline lysis method of plasmid isolation was originally developed by ____ and ____ (1979).
Brinboim and Doly
Type of method of plasmid DNA:
A suspension of bacteria is made in isotonic solution which is subsequently subjected to lysis by an alkaline solution containing a _____ and _____.
detergent (SDS) and NaOH
Alkaline condition denature ___ ____, ____ ____ as well as proteins.
genomic DNA, plasmid DNA
Neutralization results in _______ of plasmid and genomic DNA
Neutralization results in renaturation of plasmid and genomic DNA:
Since plasmid DNA is _____ closed, it reanneals ____ and remains in solution in ____ form while genomic DNA reanneals ____, resulting in the formation of ____.
covalently; properly; soluble
Plasmid from the supernatant can be recovered by precipitation using _____ or ______.
isopropanol or ethanol
A typical isolation procedure by alkaline lysis have the following steps.
1. Harvesting of bacteria from culture
2. Suspension of bacterial pellet in resuspension buffer
3. Lysis of bacteria
4. Neutralization of lysate
5. Clearing of lysate
6. Recovery of plasmid from cleared lysate
7. Washing of plasmid DNA
8. Elution and storage of plasmid DNA
Centrifugation is used to:
separate the bacterial cells
Clumps of bacteria in resuspension buffer leads to _____ lysis, resulting in low yield of ____.
Separating bacterial cells from the rest of the culture medium is essential as bacterial culture medium may contain ____ which can affect the enzyme activity.
To avoid such problems, a wash with _______ (pH 8.0) or ____ ____ is recommended.
Tris.EDTA; resuspension buffer
Bacterial pellet isolated from step first is _____ in resuspension buffer. Resuspension buffer contains _____,____ and _____
resuspended; Glucose, EDTA and Tris-HCl.
Glucose is required to make the solution _____.
EDTA ____ the divalent cations which are released upon bacterial lysis.
Divalent cations are required for many ____ ____.
EDTA action results in ____ of many enzymes which may harm plasmid DNA.
Tris.Cl acts as a ____ _____
Lysis of bacteria:
In this step, bacterial suspension is treated with lysis solution which contains ____ ____ ____ (SDS) and ____
Sodium Dodecyl sulfate (SDS) and NaO
SDS is a detergent which solubilizes the ____ and denatures ____ components of the cell membrane, leading to lysis and release of the cell contents.
High alkaline condition due to NaOH denatures the ____ and ____DNA.
plasmid and genomic
Neutralization of lysate:
Addition of neutralization solution (acidic potassium acetate) brings the ____ ___ back to normal, resulting in precipitation of ____ and _____ DNA
lysate pH; protein; genomic
Both plasmid and genomic DNA renatures upon addition of ____ buffer.
Plasmid DNA renatures in its correct conformation due to its ____ and ____ nature and therefore, remains in the solution.
Genomic DNA ____ due to ____ ___of both the strands and its association with the bacterial cell wall.
circular and covalent
precipitates; random association
Clearing of lysate:
Precipitate formed upon addition of neutralization solution is separated by aqueous solution by _______ .
Recovery of plasmid from cleared lysate:
Cleared lysate contains ____. Most protocols use precipitation to recover plasmid from solution. Plasmid DNA can be precipitated by either addition two volumes of ____ ____ or 0.7 volume of _____ to the cleared lysate. Centrifugation at high speed results in collection of plasmid in a form of ____.
Alternatively, DNA can be recovered by binding to ____ under high salt conditions.
Precipitation of plasmid DNA contains salts which need to be removed from the plasmid. For this purpose ____ ____ wash is given to the pellet.
8. Storage of Plasmid:
The plasmid pellet can be dissolved in double ___ __ or ____ (pH 8.0).
distilled water or TE
Prep Kit is called:
Qiagen Miniprep Kit
Plasmid preparation (prep) is comprised of 3 basic stages:
-Preparation and clearing of a ____ ____
-____ of DNA onto the QIAprep (silica gel) membrane
-____ and elution of DNA
DNA/RNA absorb ultraviolet (UV) light at a wavelength of ____
The Beers-Lambert Law relates the ____ ____ to ___ ___
UV absorption to DNA concentration
The more UV light absorbed, the higher the concentration of ___ _____
An optical density (or "OD") of 1 corresponds to a concentration of ___ μg/ml for double-stranded DNA. Valid for up to an OD of __-
A measure of sample purity is the _____ ratio.
Measures protein contamination
Pure DNA yields a ratio of ___(RNA ~2)
Beers law absorbance=
e x conc x I
lab 5: DNA RESTRICTION DIGEST AND GEL ELECTROPHORESIS
In nature, restriction enzymes are found in ____ and archaea and serve to restrict viral growth by ____ ____ _____
destroying foreign DNA
Restriction enzymes usually occur in combination with one or two ____ enzymes (DNA methyltransferases) that ___ the cell's own ___ from cleavage.
Type II enzymes cut DNA at defined positions close to or within their ____ ____ and are commonly used in the laboratory. There are over ten subtypes with different types of ____ ____, ____ ____ and ____ ___
recognition sites, cleavage sites and cofactor requirements.
The most common Type II enzymes cleave within their ____ ____ (e.g., BamHI, EcoRI); sites can be ____ or ____
recognition site; symmetric or asymmetric.
The each DNA strand shows ____
The two strands comprising dsDNA are oriented in an ____ manner.
HaeIII--> cleavage site is
EcoRI and HindIII are ____ sites
restriction endonucleases are enzymes that cleave DNA at specific ____ ____. Sequences are ___-___ pairs long
nucleotide sequences; 4-6
because the same recognition sequence in both strands of the DNA duplex, the restriction endonucleases can bind to and cleave ___ __ of DNA
Single stranded DNA will pair, and then be joined by ____ ___
Working with ____ ____.
Plasmids were found mainly in ____.
Plasmids are ___-____ DNA and are usually about ____-_____ base pairs in size. Plasmids are self replicating and they replicate independently of the bacterial chromosome. You will do a ___ ___ and ___ ___
1,000 - 4,00
single digest and a double digest.
The endonucleases being used are ____ and ____ . You will also make a ____ ____ of your total chromosomal DNA which was extracted previously from the same line of E. coli.
EcoRI and PstI
Use fresh tip for every new component. This will allow you to avoid ____-_____
The loading dye, ____ ____, is used to track the progress of the sample. This contains ____ which weights the sample so that is sinks into the well during loading.
Bromophenol Blue; glycerol
When loading just break the ____ of the buffer over the well with the micropipette tip and slowly inject the sample. The sample should sink into the well.
Now turn on the power to about ___ volts. If voltage too high, your gel will ____ and agarose will ____. At lower voltage electrophoresis will go ___.
110; overheat; melt
Observe for bubbles arising from electrodes in the gel buffer. This indicates that the current is ____
DNA fragments are ____ charged. So, fragments will move from ____ to ____
negatively; negative to positive
The larger the DNA fragment, the ___ it will move through the gel matrix.
The DNA fragments are stained by the chemical dye ______ ____. This binds to DNA by _____ (fitting in) between the bases. The dye is added is a component of the agarose gel.
The stain Ethidium Bromide is _____ and should be handled with care.
The gel will be placed by your instructor on a ____ ___ and the ___ ____DNA will fluoresce under UV light
UV illuminator; EtBr-stained
LAB 6: Bacterial Transformation
Transformation is the genetic alteration of a cell resulting from the direct uptake, incorporation and expression of ____ ___ from its surroundings and taken up through the cell membrane(s).
Bacterial transformation was first demonstrated by _____ _____ in 1928.
Transformation occurs naturally in some species of bacteria, but it can also be effected by ____ ____in other cells.
For transformation to happen, bacteria must be in a state of competence: i.e., possess the capacity to take up ____ ___ ____
naked extracellular DNA.
One basic cloning technique begins with the insertion of a ___ _____ into a ____ ______ (E. coli and its plasmids are commonly used) with the use of ____ enzymes.
_____ is a general term describing the uptake and expression of foreign DNA in a cell
First, a foreign gene is inserted into a ____ ___ to produce a recombinant DNA molecule. The plasmid is inserted to a bacterial cell by ____, producing a recombinant bacterium, which reproduces to form a clone of identical cells.
bacterial plasmid; transformation
Every time the bacterium reproduces, the recombinant plasmid replicates as well, but not ____.
The potential uses of cloned genes fall into two general categories:
-First, the goal may be to produce a protein product.
-Alternatively, the goal may be to prepare many copies of the gene itself.
Most protein-coding genes exist in only one copy per ____, so the ability to clone rare DNA fragments is very valuable
Cells that have been treated for transformation are called _____ ____
The preparation involves treating cells with an ice-cold solution of ____ ____ as a source of _____ cations to disrupt the ____ ___ ___ and ____ by creating ____ through which foreign DNA can enter.
bacterial cell wall and membrane
Cations neutralize the ____charges of _____ in the cell membrane and the DNA entering the cell.
Heat shock the cells at 37-42ºC so the ____ ____ goes into the cell and the openings of the cell wall and membrane become _____.
recombinant DNA; closed
To determine which cells have become competent and obtain the recombinant plasmid DNA molecule, they are grown in a liquid medium, and then transferred to an a____ ____ containing the antibiotic (selection ingredient) to which the recombinant bacteria should be ___
Only the cells transformed with the gene of interest and the antibiotic-resistant gene will grow to produce ____.
If a virus is used to transform a cell, it is called _____.
To describe mammalian transformation the term used is ____.
A plant or animal, whose cells have genetically altered by transformation, is called ____.
Another common technique alternative to calcium chloride-mediated transformation of bacteria is _______. In this case the special instrument produces briefs ____ to introduce recombinant DNA into the cells without ____ most of them.
electroporation; electroshocks; killing
Exposure E. coli cells to CaCl2 allow for ____ ___ uptake as cells become competent. Plasmid DNA adheres to receptor sites on the ______ cell and then enters the cell.
plasmid DNA; bacterial
Cells are heat shocked to allow for increased efficiency by which ____ ____ enters the cells
Cells are then grown on nonselective medium to allow for expression of the _____-____gene marker and then placed on selective medium to allow for identification of plasmid-containing cells that develop into colonies on selective medium.
Rapidly transferring cells back to the ice bucket for 2 minutes. This is done to ____ the pores of the cell membrane
LB plates contain _____
-Extreme cold causes ____ to appear in bacterial membrane. ___ DNA can move through this
-Bacteria DNA is ____
-When bacteria are heated, pores are ___.
-Bacteria is heated again so that the ____ that entered DNA, stays inside. These are called ____ ____.
closed; transformed bacteria
Summary Part 2:
-The _____ bacteria (the ones with no plasmid) is filtered out by growing all of the bacteria in an ____-___ medium
-____ bacteria is killed by the ____ in the medium because they don't have the ____ ___ gene
On the plate, you will see the ____ ____ grow
untransformed; antibiotic medium
Untransformed; antibiotic resistant
LAB 7: DNA Fingerprinting
____ ___ ___ ___:
A molecular method of genetic analysis that allows individuals to be identified based on unique patterns of ___ ___ ___ in specific regions of DNA.
Restriction Fragment Length Polymorphisms (RFLPs)
-restriction enzyme cutting
The technique takes advantage of the _____ in individual genetic codes. Even though all members of a species have essentially the same genetic makeup, these slight differences account for variations in _____ between individuals.
RFLP methodology involves cutting a particular region of DNA with known ____, with ____ ___, then separating the DNA fragments by agarose gel _____ and determining the number of fragments and relative sizes. The pattern of _____ will differ for each individual tested.
variability; restriction enzymes
electrophoresiS; fragment sizes
RFLP is one technique used by forensic scientists in ____ _____. It is also used for ____ ____, studying evolution and migration of wildlife, and detection and _____ of certain diseases.
tracing ancestry; diagnosis
Most RFLP markers are ____-____ (both alleles in heterozygous sample will be detected) and highly _____-____
Prenatal diagnosis of sickle-cell anemia is performed by Southern blot analysis to detect a restriction fragment length polymorphism (RFLP) in the ___-____ gene
Fill electrophoresis with ____ buffer
DNA consists of a series of ____ base molecules held together by weak ____ -____.
molecular scissors or "cutting" enzymes are
LAB 8: Ames Test
A ____ is a permanent alteration in DNA sequence
Mutations may be:
-_____-____ pair substitutions
_____ or -_____ of
one or more base pairs
major alteration in ____ ____-
deletion or insertion
____ mutations happen naturally and randomly and are usually linked to normal biological or chemical processes in the organism
Induced mutations result from the influence of an extraneous factor, either natural or artificial:
Natural and synthetic chemical
The Ames test uses a number of different strains of _____ _____ selected for their ability to reveal the presence of specific types of _____ . Genes involved in _____ _____
The Ames test is used extensively during the development of _____ and ____ chemical compounds
industrial and pharmaceutical
Many known carcinogens have been shown by the Ames test to be strong ____
Somatic mutations occur in any cell except ____ ____ and are not heritable
Germ-line mutations occur in ____ and are inherited
____ mutations occur within genes located on the autosomes, whereas X-linked and Y-linked mutations occur within genes located on the ___ and ___ ____, respectively
Autosomal; X and Y chromosome
___ ___ or base substitution is a change of one base pair to another
_____ _____result from insertions or deletions of a base pair
____ ___ interrupt an essential process and result in death
The expression of ____ _____ depends on the environment in which the organism finds itself
The ____ ____ is a biological assay to assess the mutagenic potential of chemical compounds.
As cancer is often linked to DNA damage, the test also serves as a quick assay to estimate the _____ _____ of a compound since it is difficult to ascertain whether standard carcinogen assays on rodents were successful.
The tester strains are specially constructed to have both ____ and ___ ____ in the genes required to synthesize ____, which allows for the detection of mutagens acting via different mechanisms.
frameshift and point mutations; histidine
The tester strains also carry mutations in the genes responsible for _______ synthesis, making the cell wall of the bacteria more _____, and in the _____ _____ system to make the test more sensitive.
Rat liver extract is optionally added to simulate the effect of ____, as some compounds, like benzo[a]pyrene, are not mutagenic themselves but their ____ ____ are.
metabolism; metabolic products
The bacteria are spread on an agar plate with a small amount of ____. This allows the bacteria to grow for an ____ ___ and have the opportunity to mutate.
histidine; initial time
When the histidine is depleted, only bacteria that have mutated to gain the ability to produce its own ____ will survive. The plate is incubated for 48 hours. The mutagenicity of a substance is ____ to the number of colonies observed
Drugs that contain the _____ ____sometimes come back positive for Ames when they are indeed safe. ____ is an example that gives a positive Ames yet is still used in treatment today.
The conditions of the Ames test are dosed at very high concentrations and with nitrate compounds that can potentially generate ____ ___-, an important signal molecule, will give a false positive.
nitric oxide (NO)
The original test also doesn't count for metabolites that are formed in the ____ ____. Modified tests can include ____ ___ fraction to help recreate the system and observe whether the parent molecule's metabolites formed in the hepatic system are positive.
hepatic system; liver S9
A ____ ____ can occur either in a protein-coding region or in any part of the genome
_____: loss of one of the nitrogenous bases (usually a purine) in an intact double-helical DNA molecule
Mutagens act in a variety of ways but they all have the
ability to alter the ____ __ ___ (e.g. recall point mutations, frameshift mutations, etc.)
within the genome.
DNA base sequence
The procedure you will perform was developed by Bruce Ames in order to simply,
quickly and cheaply ____ ____ (mainly commercial products) for their ability to act as
mutagens in bacteria
These strains of S. typhimurium used are known as _____.
strain is defined as an auxotroph if it is unable to produce a ____ ____ (the test organism
in this experiment cannot synthesize the amino acid _____) and thus will not grow unless the
nutrient is supplied in growth media.
required nutrient; histidine
Auxotrophs are usually produced as a result of a
mutation(s) that occurs in a ____ (a bacteria that is able to synthesize the particular
The mutant S. typhimurium strains are ____ auxotrophs
due to a mutation in a gene encoding an enzyme required for histidine
These S. typhimurium strains also have other characteristics that enhance their
ability to ___ ___
histidine; detect mutations
A mutation distinct from that present in the histidine biosynthesis
gene makes the cell wall of the bacteria more permeable to ____ ___ that normally would
not enter the cells.
They also possess a mutation in a gene responsible for ____ ____ and ____ of ___ ___, thus increasing their sensitivity to mutagens.
This last mutation also
disrupts the gene for ____, so these strains must be supplied with biotin as well as histidine.
proper excision and
repair of DNA damage
The test substance diffuses into the surrounding media and, if mutagenic, will induce ____-___ which will then allow
grow into visible colonies. The more _____observed near the test substance, relative to
experimental controls, the more mutagenic the substance is likely to be.
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