Restriction Enzyme Digestion and Ligation

Cloning by restriction enzyme digestion and ligation is a simple and easy way of moving a fragment of double stranded DNA from one plasmid to another.

Start by:


1.  Choosing restriction enzymes for which the recognition sequences flank your gene of interest
2.  Incubate the reaction for the recommended amount of time
3.  Purify your fragment
4.  Ligate into your plasmid of interest

Restriction Enzymes

Restriction enzymes (RE) function by cutting double stranded DNA at specific 4 to 8 base pair inverted repeat recognition sequences within the target DNA.  The products of DNA cleavage are either blunt ended or contain 5’ or 3’ overhangs.  

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Agarose Gel Extraction

Most restriction enzymes can be heat inactivated after the digestion is complete however some researchers prefer to purify the DNA fragment before moving into the ligation step. Purification of the fragment occurs by separating the digested fragment of interest on an agarose gel and then the DNA is purified from the agarose.

DNA Ligation

Regardless of the type of end generated by restriction digestion cleavage of the DNA results in fragments with 3’-hydroxyl groups and 5’-phosphate groups.  DNA ligase covalently attaches the 5’-phosphate to the 3’-hydroxyl in an ATP dependent reaction, joining two fragments of DNA.

Enzymes for DNA modification

DNA will sometimes need to be modified in order to get successful cloning. Possible modifications can include dephosphorylation (CIAP), filling in of uneven DNA ends (Klenow), etc.

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