Assembly of VH and VK gene fragments with linker DNA

GeneAmp dNTPs (see Protocol 5) NuSieve GTG agarose (see Protocol 5) 50 X TAE buffer (see Protocd 2) WTzard PCR Preps DNA Purification System (see Protocol 5) 

Estimate the quantities of VH and VL DNA prepared by the primary PCR reactions (Protocol 5) and the quantity of linker DNA prepared in Protocol 6, using a dot spot technique. Adjust the concentratkms of VH and VL PCR products to 5 ng/nJ and that of linker DNA to 2 ng/pL 

Set up two sets of jumping PCR reactions, the first for the VH products involving nine reactions (one for each trf the nine primary PCR reactions from Protocd 5) and the second for the VK products involving eight reactions (one for each of the ei t primaiy PCR reactions from Protocol 5). For each of the 17 jumping PCR reactions, set up the following 100 pi mixture  

Overlay with 100 pi of mineral oil and place the tubes in a DNA thermal cyder preset at 94 °C. 

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

Return the tubes to the thermal cycler pre-set at 94 C and immediately cycle at 94 C for 1 min. 55 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 for 10 min before cooling to 4 C (storage). Separate each PCR product by electrophoresis on a 3.0% (w/v) NuSieve GTG agarose gel ( 5 mm thick), containing 0.5 icl/ml ethidium bromide, prepared In 1 x TAB buffer alongside 1, 1 of 1 kb PLUS DNA ladder. 

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