Restriction enzyme digestion of assembled scFv

ssembled scFv (from Protocol 9 is digested with S/il (located at the 5 end of the heavy chain) and. Notl (located 3 of the light chain) restriction enzymes to allow cloning into the phagemid vector pHEN-1 cut with the same two enzymes (.see Protocol 12). For mouse antibody scFv, S/il and Notl sites are used because there are extremelv rare cutters. [Pg.44]

Estimate the quantities of assembled scFv purified ftom Protocol 9 using a d( spot technique. [Pg.45]

Mix gently by pipetting the reaction mixture up and down. Follow with a quick ( touch ) spin-down in a microcentrifuge. [Pg.45]

Overlay the mix with mineral oil to prevent evaporation. Incubate at 50 C for at least 12 h or overnight. [Pg.45]

Extract with phenot chloroform i oamyl alcohol, precipitate with ethanol, wash, and dry. Resuspend in 42.5 p,l of water, [Pg.45]

Phenolxhloroformiisoamyl alcohol (25 24 1, by vol.) (see Protocol 9 NuSieve GTG agarose (see Protocol 5) 50 X TAE buffer (see Protocol 2) Wizard PCR Preps DNA Purification System (see Protocol 5) [Pg.45]

Add the following components in the order stated to a 1.5 ml microcentrifuge tube [Pg.45]

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