Restriction rare cutters

Use of pulsed-field gel electrophoresis (PFGE) to separate large DNA fragments Permits isolation of large DNA fragments obtained by use of restriction endonucleases (rare cutters) that result in very limited cutting of DNA. 

Restriction Endonucleases for Rare Cutter Jumping Libraries... 

The series of tag plasmids used by us in rare cutter jumping has been constructed from suppressor-containing plasmids with polylinkers (22) by introduction and deletion of restriction sites. The polylinker sites of currently available plasmids and a corresponding list of enzymes potentially usable as primary cleavage enzymes are listed in Table 2. 

The assembled scFv (from Protocol 9) is digested with Sfil (located at the 5 end of the heavy chain) and Notl (located 3 of the fight chain) restriction enzymes to allow cloning into the phagemid vector pHEN-1 cut with the same two en ymes (see Protocol 12). For mouse antibod[y scFv, Sfil and Notl sites are used because there are extremely rare cutters. 

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