Assembly of single chain Fv antibody fragments

Estimate the quantities of VH-linker and linker-VL DNA prepared by the jumping PCR reactions (Protocol 7) using a dot spot technique. Adjust the concentrations of VH-linker and linker-VL PCR products to 5 ng/jcl. 

Overlay vrith 100 pi of mineral ofr and place the tubes in a DNA thermal cycler preset at 94 °C (Hot Start). 

5 pi of AmpliTaq DNA polymerase to each reaction toieath the mineral oil, using separate pipette tips for each addition. 

Return the tubes to the thermal cycler pre-set at 94 and immediately cycle at 94 °C for 1.5 min, 69 C for 1 min, and 72 °C for 2 min over 25 cycles. Follow the last cycle with a final extension step at 72 °C fbr 10 min before cooling to 4°C (storage). Separate each PCR product by electrophoresis on a 2.5% (w/v) NuSieve GTG agarose gel ( 5 mm thick), containing 0.5 pl/ml ethidium bromide, prepared in 1 X TAE buffer alongside 1 pi of 1 kb PLUS DNA ladder. 

Visualize the bands on a UV light box, cover the screen with Saran Wrap to prevent nucleic acid cross-contamination. 

In the final assembly reaction, the VH-link and link-VK DNA are joined into a single chain Fv antibody fragment and restriction sites are added. These restriction sites are used to done the scFv into a phagemid vector as a S/il-Notl DNA fragment. The primers contain either S/il (Mouse VH Back Sjif primers) or Not  

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