20 Life Technologies
™
| Vector Construction and Preparation
How do I check for orientation of my gene of interest after using blunt-
end fragment cloning?
The simplest way to check for the orientation of a fragment cloned
using blunt-end ligation is to generate an asymmetric cut in the
inserted fragment and a single or double cut in the vector. The
asymmetric cut in the gene of interest should be close either to the
5' or 3' end of the gene so that the resulting digestion fragment can
readily be resolved on an agarose gel to establish the orientation
of your gene. As a control, digest the empty vector with the same
restriction enzymes.
Do you have any more detail about the bacterial origin of
replication on pCHO 1.0?
The bacterial origin of replication on pCHO is derived from the mutated
pMB1 ori from pUC19, and confers replication at high copy number.
Do I need to test my construct if the insert expressed well in
another vector?
Successful expression in another cell line and/or vector does not
guarantee expression or secretion when placing sequences in the
pCHO/Freedom
®
CHO-S
®
system. It is highly recommended that
pCHO constructs be tested by transient transfection in CHO-S
®
cells
before proceeding with selection.
Vector Construction and Preparation
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