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78 Cards in this Set
- Front
- Back
The sequences on MCS recognize _____.
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The sequences on MCS recognize many different RESTRICTION ENZYMES.
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The beta-galactosidase gene is also called _____.
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lacZ gene
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Plasmids can be designed to make copies of _____.
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- DNA
- mRNA - protein |
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procedure for discarding recombinant e. coli cells
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- liquids: bacterial waste flask
- solids: biohazard bags |
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Ca++ ions
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Required for CLASSICAL PATHWAY to function
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The MCS serves as the site for _____.
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The MCS serves as the site for DNA INSERTION (and is opened by restriction enzymes).
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The MCS occurs _____.
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The MCS occurs in the middle of the lacZ gene.
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Haemophilus influenzae & Hind II
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The first restriction endonuclease enzyme, called Hind II, was isolated from Haemophilus influenzae. It recognized only one specific 6 bp dsDNA sequence.
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parameters that effect how fast molecules move through the gel during electrophoresis
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- agarose concentration
- voltage applied to gel - shape of the DNA (linear, circular) - molecular size of DNA fragment |
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Mg++
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Required for ALTERNATIVE PATHWAY to function
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competence factors
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1. The phase of the bacterial growth cycle at the time of collection"
- competence develops as culture ceases exponential growth - competency common in eary stationary phase, but only small portion actually becomes competent 2. The media in which the cells are grown: - nutritional conditions may be a limiting factor, causing competence |
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vector
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DNA molecule designed to carry a cloned DNA fragment
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Using Bacterial Plasmids to Clone Genes
Put the following events in chronological order: - transformation - use of enzyme ligase - use of restriction endonuclease - reproduction of bacteria |
1. RESTRICTION ENDONUCLEASES are used to cut the plasmid and isolate the DNA source.
2. ENZYME LIGASE is used to incubate the fragments together in a ligation reaction. 3. The products of the ligation reaction are TRANSFORMED into competent bacteria cells. 4. The transformed bacteria REPRODUCE. |
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three commonly used vectors
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- plasmids
- phages - artificial chromosomes |
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ligation reaction
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Many copies of the cut plasmid fragments and cut source DNA fragment are incubated together with the enzyme LIGASE.
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Plasmids can be designed to make copies of _____.
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- DNA
- mRNA - protein |
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DNA standard curve x-axis
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DISTANCE migrated from well
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erythrocytes
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red blood cells
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stop buffer
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- added to mixture after incubation of 30 minutes
- phosphate buffered saline with EDTA that chelates cations such as Ca++ and Mg++ so they are not available |
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Easiest and most commonly used vector type.
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plasmids
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Ligase forms _____ bonds between the ends of the plasmid and DNA insert.
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Ligase forms PHOSPHODIESTER bonds between the ends of the plasmid and DNA insert.
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TRUE/FALSE
Enzymes that recognize the MCS are usually very specific and will not cut the plasmid at other sites. |
TRUE
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recognition sequence
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Also restriction site, it is the sequence on the DNA that is specific for its matching restriction endonuclease enzyme.
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DNA standard curve y-axis
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Log base 10 of DNA fragment SIZE
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white blood cells
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- neutrophils
- lymphocytes - monocytes - eosinophils - basophils |
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histones
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- exclusively prokaryotic
- proteins found on chromosomes |
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conjugation
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direct cell-cell contact and DNA exchange via pili formation
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Disadvantage of using plasmids as cloning vector.
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DNA fragment must be relatively small (<15 kb)
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After the ligation reaction, the products are transformed into _____ bacterial cells.
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The products transform into COMPETENT cells.
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lacZ gene
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- part of the lac operon
- produces enzyme beta-galactosidase |
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methylation of DNA
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Methylation of DNA prevents the restriction enzymes from cutting the recognition sequence on the enzyme's own sequence on the bacterial DNA.
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P-20
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The name of the micropipettor used that holds up to 20 MICROLITERS.
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components found in a blood sample
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- RBCs
- WBCs - plasma - platelets |
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organelles exclusive to eukaryotes
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- Golgi apparatus
- mitochondria - chloroplasts - endoplasmic reticulum - nuclear envelope |
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transformation
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- ability to take in an incorporate exogenous DNA into the genome
- naturally occuring in few species, must be induced in E. coli |
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transformation of B. subtilis
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- begins with reversible binding of donor DNA to surface of competent cells
- DNA is then irreversibly bound to cell and becomes insensitive to DNase which normally destroys DNA - one strand of donor dsDNA is taken in and one is degraded - remaining strand is integrated into homologous region of main chromosome |
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Plasmids replicate [independently] or [dependently] from the main bacterial chromosome.
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independently
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Competent cells can take up _____.
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Competent cells can take up EXOGENOUS DNA from their surroundings such as plasmid DNA.
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What is the effect of combining X-gal and beta-galactosidase?
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X-gal causes beta-galactosidase to produce a blue dye making the bacteria look blue.
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ethidium bromide
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An intercalating dye that, when present, causes DNA to flouresce a bright orange-pink color.
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MBL
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Mannan-Binding Lectin Pathway:
Initiates complement activation after MBL binds to certain encapsulated bacteria. |
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blood serum
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- plasma minus the clotting factors
- still contains antibbodies, proteins, sugars, etc. |
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organelles in prokaryotes
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- ribosomes (also in eukaryotes)
- cell walls - capsules - inclusion bodies - gas vacuoles - flagella |
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naked DNA
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DNA with no surrounding cell structures
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site specific recombination
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recombination that requires integration into homologous regions
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______ binds to the plasmid's origin for DNA replication (ori).
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DNA polymerase
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P+ = Reproduction rate of bacteria containing plasmids.
P- = Reproduction rate of bacteria containing no plasmids. Relate the two variables: P+ ___ P- (< , > , =) |
P+ < P-
Bacteria with plasmids take longer to generate because plasmid DNA requires the same "building block" molecules as the main chromosome. |
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In the gel electrophoresis lab, what is the difference between a blue bacterial colony and a white colony?
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BLUE contains:
- plasmid - NO DNA insert WHITE contains: - plasmid - DNA insert (correct/maybe incorrect) |
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Southern transfer
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DNA fragments in the agarose gel are transferred to a nitrocellulose membrane for permanent storage.
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Alternative Pathway
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- activates the complement cascade
- does not require presence of antibody - pathogen's surface molecules initiate activation directly |
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method of extracting serum
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- collect blood without an anticoagulant
- let it sit until it clots - centrifuge the sample - the liquid is serum |
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operon
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a set of genes with related functions that are transcribed as one mRNA from one promoter
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Plasmids often carry genes for resistance to __________.
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antibiotics
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Plasmid DNA is added to an agar plate containing a strain of non-competent E. coli cells.
Will transformation occur? Why or why not? |
NO. Only competent cells can take in exogenous DNA such as the plasmid DNA in this example. Therefore, transformation will not occur.
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In the gel electrophoresis lab, you've separated the blue colonies from the white colonies. Do all the white colonies contain a DNA insert?
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YES. The white colonies carry a DNA insert, although not necessarily the correct one.
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Northern transfer
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RNA fragments are separated by gel electrophoresis then transferred from the gel to a membrane for storage.
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Classical Pathway
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- activates complement system
- initiated b y binding of antibody to antigen on pathogen cell surface - requires prior specific immunity to create the specific antibody needed |
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visual sign of RBC lysis
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the normally clear, golden plasma or serum becomes a pink or reddish color due to the presence of free hemoglobin
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binary fission
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bacterial reproduction mechanism
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transformation of B. subtilis
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- begins with reversible binding of donor DNA to surface of competent cells
- DNA is then irreversibly bound to cell and becomes insensitive to DNase which normally destroys DNA - one strand of donor dsDNA is taken in and one is degraded - remaining strand is integrated into homologous region of main chromosome |
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TRUE/FALSE
Most of the plasmids used for cloning produce very few copies of the plasmid per cell. |
FALSE
Most of the plasmids used for cloning produce MANY (tens or hundreds) of copies of the plasmid per cell. |
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pGEM
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plasmid carrying a gene for ampicillin resistance
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Why don't the white colonies produce a blue dye even when X-gal is added?
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The blue dye requires both beta-galactosidase AND X-gal, and beta-galactosidase isn't transcribed in the white colonies.
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visibility of rRNA, tRNA, & mRNA on agarose gel
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- rRNA molecules can be seen
- tRNA and mRNA rarely seen without using probes |
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MAC
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membrane attack complex
- perforates target cell membrane - causes lysis of target cell |
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Gal-3G
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- sugar molecule
- surface antigen found on rabbit RBC - human blood contains antibody vs. it |
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generation time
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the time required for the population to double in size
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required treatment for transformation of E. coli
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treatment with calcium chloride to make cell membranes more permeable
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competence
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the physiological state of cells when they can physically bind homologous DNA to their surface and take it inside
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auxotrophs
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mutants that grow only on media that contains the essential yet missing molcules
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Name three types of DNA sequences commonly found on plasmids used as cloning vectors.
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- MCS for enzyme recognition
- gene marker to identify bacterial cells lacking the plasmid - DNA insert recognition |
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You are color-blind. You test a certain colony of transformed E. coli and discover that its DNA does not contain the lacZ gene sequence. Predict the color of this bacterial colony after adding X-gal.
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WHITE. The lacZ gene transcribes beta-galactosidase, which reacts with X-gal to produce the blue color.
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MCS stands for _____.
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multiple cloning site
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You have just isolated a certain DNA fragment from a mouse and you'd like to clone it for further study using plasmids. Should you choose plasmids with antibiotic resistance? Why?
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YES. Cloning plasmids usually carry an antibiotic resistance gene. This resistance will allow you to separate the transformed cells from those that haven't successfully transformed.
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expression vector
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plasmids that are engineered to produce a large quantity of one cloned protein
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complement system
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- approx. 20 blood proteins
- sequentially activated only during infection - activation forms MAC |
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sialic acid residue
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- found on sheep RBC membranes
- sugar molecule - binds to Factor H in human circulatory system which blocks the alternative pathway |
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transduction
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bacterial DNA transfer via phages
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