Construction
of expression vector (when requesting for vector design):
Preparation of interest DNA: Genomic DNA
or cDNA conditions
Accurate information and sequence concerning genes:
What kind of gene?
What is the source of the gene? (e.g., mouse gene,
human gene, etc.)
Determining what kind of promoter will be used for gene
expression: Ubiquitous promoter or tissue specific promoter
Purification of expression vector
:
Completed vector: Prepare 20ug or more of
Plasmid DNA with concentration of 500ng/ul or more. When
preparing Plasmid DNA, use a kit (midi or maxi prep. kit,
etc.) or purify it through phenol extraction.
Identification of the founder: Preparing
the primer for TG screening:
Detect an insert gene; product size should
be 500bp~1kb.
If the insert gene was derived from a mouse, a specific
design should be prepared such that the endogenous gene
cannot be detected.
The primer should be about 20mer long with Tm value
of about 60 degrees.
