Question

Cloning a DNA Fragment to a Bacterially-Derived Plasmid Vector”

“Table 1: Fragment Lengths”

DNA TYPE: LONGEST LENGTH

FOREIGN: 716

PLASMID: 2837

Post-Lab Questions

1. What is the expected size of the plasmid plus the cut foreign DNA?

2. What type of ends to the enzymes BamHI and EcoRI produce? How does this type of end facilitate cloning?

3. What enzyme is necessary to permanently link the digested foreign and plasmid DNA together to form the recombinant DNA molecule? How does this enzyme work?

4. How would you clone a gene into a plasmid if there were no common restriction sites between the two DNA sequences?

Answer 1

1. The expected size of plasmid plus cut foreign DNA is --> plasmid size + foreign DNA size + restriction enzyme cut sequence length

i.e., 2837 + 716 + 4 to 6 = 3557 to 3559 approximately.

2. Both BamHI and EcoRI produces sticky ends. Sticky ends are more useful in cloning because they ensure that foreign DNA is inserted in right direction into the plasmid.

3. The enzyme that is necessary to permanently ligate the foreign DNA and plasmid DNA together to form the recombinant DNA molecule is DNA ligase.

It carries out  by catalyzing the formation of an inter-nucleotide ester bond between phosphate and Deoxyribose of foreign DNA and plasmid DNA.

4. There are many cloning methods that do not require restriction enzymes or ligases. The technique of producing "customized cohesive ends" by a combination of PCR primer design and lambda exonuclease digestion cloning can be done with out using common restriction enzymes