Why it is important for a plasmid to have a \'selection marker\' such as amp^R (
ID: 82930 • Letter: W
Question
Why it is important for a plasmid to have a 'selection marker' such as amp^R (ampicillin resistant gene)? What are the roles of resuspension buffer, lysis solution and neutralization buffer? Also study their compositions, role of each reagents (e.g., NaOH, SDS, potassium acetate etc.) in these solutions. How DNA molecules move through gel? and towards what direction? What is the dye used to stain DNA/RNA on gel? Why does the plasmid stick to the column while the residual contaminants wash out as flow through? How does alcohol help purifying plasmid DNA from other contaminants (salts)? Why plasmid is not soluble in alcohol, but soluble in water? Lab 9: Restriction mapping Define restriction site and restriction enzyme? How does restriction enzyme recognize its target (restriction) site? What do you understand by cohesive ended- and blunt ended DNA fragments? How do different DNA fragments that are obtained after restriction digestion help you to construct a restriction mapping? What is the DNA length measurement unit? Now practice the following restriction mapping problems.Explanation / Answer
Lab 9: Restriction mapping
Answer 1: Restriction enzymes can be defined as the enzymes that can cut the DNAat specific sites. These sites are known as restriction sites which are specific for a particular restriction enzyme. Restriction enzymes can be divided into different categories depending upon the the structure and cutting of the restriction site. This gives a definition of restriction site as being the site of the the DNA sequence where the restriction enzyme would cut the DNA.
Answer 3: sticky or cohesive ended DNA fragments are those have protruding single single stranded overhangs that are in the form of unpaired nucleotides that can anneal or join onto one more complementary sequence that would base pair with this overhang.
Blunt ended DNA fragments do not contain such overhangs that would anneal to the complementary single stranded nucleotides readily. Thus, the blunt end ligation is less efficient comparatively. These are joined by DNA ligase that form covalent Bonds between adjacent nucleotides.
Answer 4: the digested samples obtained from restriction enzymes are subjected to gel electrophoresis for separation and subsequently, the sizes are recorded. Following this, each restriction site will be mapped according to the size of the digests and this information can be used to map the digests correctly on the mapping sites.
Answer 5: the measurement unit of DNA or RNA is kilobase - kb . One kb = 1000 bp.
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