DNA Science

Restriction Digest• Restriction Digestion is the

process of cutting DNA molecules into smaller pieces with special enzymes called Restriction Enzymes (RE)

• Restriction Digests begin by mixing the DNA and the RE.

• RE are kept on ice until the procedure begins (Why?)

• Variables (pH, NaCl concentrations, etc) are optimized by adding a specific buffer for the enzyme being used.

Restriction Digest• Once all the ingredients are

mixed in the reaction tube, the tube is incubated at the Restriction Enzyme's optimal temperature for 1 hour or longer.

• Sample is ready for Gel Electrophoresis or DNA Ligation.

• Restriction Enzyme Animation• Lab Overview Animation

Ligation• The final step in the construction

of a recombinant plasmid is connecting the insert DNA (gene or fragment of interest) into a compatibly digested vector backbone.

• This is accomplished by covalently connecting the sugar backbone of the two DNA fragments.

• This reaction, called ligation, is performed by the T4 DNA ligase enzyme.

Ligation• The DNA ligase catalyzes the

formation of covalent phosphodiester linkages, which permanently join the nucleotides together.

• After ligation, the insert DNA is physically attached to the backbone and the complete plasmid can be transformed into bacterial cells for propagation.

• Ligation Animation

Transformation• Transformation is the process by

which foreign DNA is introduced into a cell.

• Before transformation, bacteria are treated with a chemical called calcium chloride, which causes water to enter into the cells and makes them swell. These swollen bacteria are then known as competent bacteria.

Transformation

• Next, plasmid DNA (containing the foreign DNA) is mixed with the competent bacteria and the solution is heated. The plasmid DNA enter the bacteria through small pores created in the cell membranes. Once in the host cell, the plasmid DNA is copied many times by the bacteria’s own DNA replicating machinery.

• DNA Transformation- Animation

Gel Electrophoresis• Gels made of agarose seaweed.• The jello consistency offers

resistance to the pieces of DNA as they try to move through the gel.

• Once the DNA samples are loaded onto the gel, an electric current is applied to the gel.

• DNA is negatively charged due to all the phosphate groups in the backbone of DNA.

• Thus, DNA will move towards the positive electrode

Gel Electrophoresis• As the pieces of DNA move

through the gel, they will meet with resistance.

• Larger pieces of DNA will have more difficulty moving through the gel than smaller fragments

• Thus, larger fragments will move slower than smaller fragments.

Gel Electrophoresis

• Gel electrophoresis• virtual lab