Q. Will extra bases in the 5' and 3' UTRs (Untranslated
regions) of my gene cloned into a transfer vector affect protein
A. For optimal protein expression, UTRs have to be
as small as possible, especially in 5' to avoid formation
of secondary structures in the mRNA. A minimal length (7-8
bases) in front of the ATG is acceptable, especially if the
Kozak consensus sequence (GCCGCCACCATGG) is included.
There are no special requirements for the 3'UTR.
Q. Is it possible to first transform the BJ5183 with pAdEasy-1
and then make this recombinant strain electrocompetent for
transformation with the linearized transfer vector?
A. Yes, it is possible. However, we do not recommend
it since BJ5183 cells support the RecA recombination system.
Our researchers have shown the occurrence of unwanted recombinations
in laboratory tests. Therefore, we recommend growth and amplification
of plasmids should not be performed in that strain of bacteria.
That is why the DH5
strain is also included in the AdenoVator kit is that
it allows amplification without rearrangements (does not support
In the following reference, researchers have first introduced
the viral plasmid into BJ5183 cells and then electroporated
the recombined transfer vector into the cells:
Zeng, M. et al. (2001). AdEasy system made easier
by selecting the viral backbone plasmid proceeding homologous
recombination. BioTechniques 31:260-262.
Q. What is the best way to screen for recombinant DNA
made with the AdEasy kit?
A. The restriction enzyme Bst XI gives a very good
diagnostic pattern to screen recombinants with the AdEasy
system. The restriction fragments of the pAdEasy-1 plasmid
are as follows:
1- 11921 bp
2- 8237 bp*
3- 5251 bp
4- 4254 bp
5- 2379 bp
6- 1399 bp
Fragment number 2 is the only one that will vary with the
size of insert and from one transfer vector to another. With
pShuttle-CMV, it shifts up to about 11-12 kb (and higher with
the insert) whereas with pShuttle, it shifts to approximately
10.5 kb. This band may sometimes be confused with band 1 but
disappearance of band 2 is a good indication of positive recombinant.
This, in conjunction with the Pac I restriction pattern, makes
identification of positive recombinants very straightforward.
Q. Upon screening recombinants with Pac I, some
clones give a 30-35 kb band and a 3.0 kb band, others the
same 30-35 kb and a 4.5 kb band. Which one is the good pattern?
A. Some recombinants might come from a recombination
event between the right side homologous regions and the origins
of replication instead of with the left homologous regions
(see applications manual). These 2 recombinants are equally
good and can very well be used but will give different restriction
patterns. In our own experience, the pattern giving a band
at 4.5 kb is generally the most abundant.