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  • Restriction enzymes identify a specific recognition sequence in the DNA, and generate double-stranded

  • breaks in the DNA duplex. Cleavage results in fragments with either a 5' or 3' overhang,

  • commonly referred to as sticky ends, or no overhang, referred to as blunt ends.

  • The 5' phosphates and 3' hydroxyls are maintained after cleavage, leaving the DNA ends ready

  • to be joined via ligation. The ability of some restriction enzymes to

  • predictably cleave DNA and generate distinct DNA bands with ligatable ends has made them

  • an invaluable tool for recombinant DNA technologies. When selecting restriction enzymes for use

  • in a cloning experiment, it is important to: determine which enzymes will produce ends

  • compatible with the selected vector confirm that recognition sites do not occur

  • within the DNA fragment to be cloned and examine the methylation sensitivity

  • of your selected enzyme to confirm that host methylation by dam and/or dcm methylases will

  • not block cleavage. NEB offers a variety of online interactive tools to aid in selecting

  • which enzyme to use, as well as parameters for setting up reactions.

  • Visit CLONEWITHNEB.com for a full list of products available for this application.

Restriction enzymes identify a specific recognition sequence in the DNA, and generate double-stranded

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B2 dna restriction cleavage methylation cloning selecting

Cloning With Restriction Enzymes

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    Cheng-Hong Liu posted on 2014/12/15
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