Lab 5 Quiz Answers
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which of the following will you complete in lab 5
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digest a plasmid with restriction enzymes transform bacteria with a plasmid streak bacteria on agar plates swab cheeks isolate DNA
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which of the following does not accurately describe an engineered plasmid?
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a dna molecule that can be extracted from bacteria using restriction enzymes
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engineered plasmids:
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-dna molecule that can confer resistance to an antibiotic -vector that can be used to insert a useful gene into a bacterial cell -circular dna molecule found in bacteria that replicated independently of bacterial chromosomal dna -dna molecule that has restriction sites
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restriction enzymes
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cut double stranded dna only at specific nucleotide sequences
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what does it mean for the cell to be competent?
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charges on the phospholipid layer are neutralized by CaCl2 to allow the cell to uptake DNA
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what does it mean that a bacterial cell has been transformed?
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a plasmid has been inserted into the cell
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agar plate storage
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-parafilmed -rubber banded together -properly labeled -placed inverted in the 37C incubator
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main purpose of repeating the three steps (denature, anneal, extend) in PCR is
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to amplify the amount of target DNA sequence
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DNA, because it has a __ charge, moves to the __ end of the field in gel electrophoresis; ____ dna molecules migrate the most quickly
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negative, positive, smaller
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wells containing the DNA samples should
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be far away from the anode so they can run to red
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determining transformation efficiency by counting the number of colonies on a lb only plate would be
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pointless because there is no selection for the ones that are transformed; no way to tell it is transformed
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bacterial transformation experiment was conducted and cells were plated on lb agar plates with and without ampicillin. both plates showed similar results; no individual colonies but lawns visible. this indicates that
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the ampicillin was bad so without selection pressure, any cell grew on the plate which formed a white lawn
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types of genes found on plasmids are
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-genes that confer resistance to antibiotic -genes that confer the ability to metabolize an unusual food source -genes that confer the ability to produce a toxin
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the types of plasmid genes that help in selection of transformants apart from normal cells are
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genes that confer resistance to antibioticc
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each pcr cycle has three main steps which repeat many times. all of the following are required for pcr process EXCEPT
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heat to denature enzymes and provide energy for the reaction
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requirements for pcr
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-specific single stranded dna primers and template dna -buffer of correct pH and concentration -heat tolerant DNA polymerase such as Taq -dNTPs and Mg2+ as cofactor
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prior to plating, what part of a transformation reaction is done on ice?
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incubation of chemically competent cells with plasmid DNA
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characteristic of touchdown pcr program
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-avoids amplification of non targeted sequences -employs initial annealing temperature above projected melting temperature of the primers -progressively lowers the annealing temperature over the course of successive cycles -simple and rapid means to amplify a range of target sequences without the need for length optimizations
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taq polymerase?
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dna polymerase that is known to stay functional at very high temps
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plasmid
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genetic structure in a cell that can replicate independently of the chromosomes, typically a small circular dna strand in the cytoplasm. they are used in the laboratory manipulation of genes
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how agarose gel is improperly cast causing it to break at the wells and become unusable?
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comb was inserted incorrectly at the adjustable end
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Given a particular plasmid has two Sma1 restriction sites and one EcoRI restriction site. This plasmid is completely digested simultaneously with the restriction enzymes EcoRI and Sma1. The digest reaction is then separated by agarose gel electrophoresis. How many bands are expected to be seen on the gel?
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3 bands
