16 Life Technologies
™
| Vector Construction and Preparation
Is there a size limitation of the total insert for pCHO 1.0? Would a large
insert like 3.0 kb insert work?
We have cloned up to 6 kb into the vector at the AvrII/BstZ17I
insertion site without any issues. Increasing vector size, in general,
compromises transfection efficiency, and therefore it is possible that a
larger insert might compromise transfection efficiency.
Is it necessary to include the 5'- and 3'-untranslated regions, or are
they already included in the vector?
Untranslated regions are not needed, as the vector supplies
all necessary regulatory elements as well as downstream
poly(A) sequences.
The User Guide suggests trying both combinations of SU1 and SU2
when subcloning two separate subunits. Is this really necessary?
For monoclonal antibodies, we typically express the heavy chain
upstream of the light chain, but we do know that both orientations work.
Vector Construction and Preparation
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