Question

1. Why must the restriction enzyme used for the digestion of TOL plasmid be inactivated immediately after the experiment? How can this be achieved?

2. Which enzyme is used for the restriction digestion of TOL plasmid?

Answer 1

1.

A plasmid that specifies the degradation of aromatic hydrocarbons. Pseudomonas putida containing the pWWO plasmid grows on toluene, m‐ and p‐xylene, m‐ethyltoluene, and 1,2,4 trimethylbenzene.

Heat Inactivation. Heat inactivation is a convenient method for stopping a restriction endonuclease reaction.

Inactivation of restriction endonucleases is generally not necessary, but in some cases it might increase the transformation efficiency.

Restriction digestion performed by incubated at 37°C for 60 minutes ( for digestion) and then at 65°C or 80°C for 20 minutes( for inactivation) .

2.

BamHl, Pstl , EcoRl, mostly present in tol plasmid and use for restriction digestion and also different toll plasmid have different restriction site present on plasmid so based on which of the restrictions site present, select Restriction enzymes for digestion

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