What are sizes of the fragments from an enzyme digestions (endonuclease) of the Lambda Phage plasmid DNA using Eco RI and Pst I? Make sure to have an uncut and double-cut reaction. Explain the mechanism of action of each enzyme and then list the fragment all sizes and draw the results.

Restriction enzymes are DNA-cutting enzymes found in bacteria.
These are site-specific endonucleases also known as molecular scissors.
These recognize and cut DNA only at a particular sequence of nucleotides.
DNA from the bacteriophage Lambda (48,502 base pairs in length) is cut with a variety of restriction enzymes and the resulting fragments are separated using gel electrophoresis.
Size of fragment of uncut lambda DNA is 48502 b.p.

Eco RI recognizes G AATTC

CTTAA G

Lambda phage has 5 recogition sites for Eco RI. It cleaves the DNA at ‘G’ of the above sequence.
Relative sites of cleavage in base pairs are 21226, 26104, 31747, 39168, 44972.
Sizes of fragments produced when the lambda DNA cut with Eco RI 21226, 7421, 5804, 5643, 4878, 3530.
Digestion of lambda DNA with EcoRI generated 6 fragments but 2 of them are so close in size that they are difficult to separate.

Pst I recognizes CTGCA G

G ACGTC

The Pst I restriction enzyme produces 29 fragments of lambda DNA.
It cleaves the DNA at ‘G’ of the above sequence.
The Pst I digest of λ DNA yields the following discrete fragments (in base pairs): 11497, 5077, 4749, 4507, 2838, 2556, 2459, 2443, 2140, 1986, 1700, 1159 1093, 805, 514, 468, 448, 339, 264, 247, 216, 211, 200, 164, 150, 94, 87, 72 and 15.
The cohesive ends (12b cos site of bacteriophage) of fragments 11501 bp and 2556 bp may anneal and form an additional band.

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