Methods

1. Cloning

1.1 Restriction and phosphatase treatment of plasmid DNA

Originally found in bacteria, endonucleases belong to a class of enzymes capable of cleaving the phosphodiester bond of DNA molecules by hydrolysis. Depending on the type they recognize site specific base sequences of 4, 6 or 8 bases respectively, which are often palindromic. Treatment of different DNA molecules with the same endonucleases allows the combination of the resulting DNA fragments in a following ligation reaction.
The treatment of DNA fragments is beneficial if the restriction was performed with only one restriction enzyme. It can also be beneficial if the restriction with two different restriction enzymes was unbalanced and single cut plasmid DNA is left. This applies in particular to the treatment of plasmid DNA with phosphatase. As the nomenclature indicates the phosphatase cleaves the phosphate group at the 5´terminus of DNA fragments and thereby minimizes the background of mono ligated plasmid DNA. Consequently the amount of transformants with insert DNA in the plasmid construct is considerably increased.
The following components were used for a basic restriction mixture:

The mixture was incubated for 10 min at 37 °C. The success of the restriction was subsequently checked in an agarose gel electrophoresis.

1.2 Restriction of PCR products:

Corresponding restriction sites were added in a PCR reaction at the 5´and 3´termini of insert DNA via modified primers. After gelextraction the following components were used for a basic restriction mixure.

Impressum/Copyright