PHEN-1 vector restriction digestion

Restriction digestion of the phage dispiay vector, pHEN-1... 

Using a 3 1 molar ratio of restriction digested pHEN-1 vector and scFv insert," set up the following reaction in a 1.5 ml microcentrifuge tube ... 

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. 

The pHEN-1 vector is first digested with Sfil restriction enzyme followed by digestion with Notl. Finally the double digested Sfil/Notl pHEN-1 vector is cut with PstI (located in the polylinker, between the Sfil and Notl sites) to reduce the vector background (i.e. rehgation of the vector on itself). 

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