This is intended to create control template preparations, in which
tail DNA is spiked with known amounts of transgene DNA to create single
copy and multiple copy standards. These are used to verify that your
Southern blot or PCR reaction is sensitive enough to detect the
integration of a single copy of a transgene. This will ensure that your
screen for transgenic founders will not miss any transgenic lines of
mice. The GTTF requires that you include and demonstrate use of these
controls in order to obtain the guarantee of 3 founders per
construct.
Assumptions:
The Haploid content of the mouse genome is 3 X 109
bp
You have 10 micrograms of DNA to spike
Transgenic founder mice are heterozygous
Size of transgene band to be detected, 2740 bp in this example
10 ug genomic DNA per lane of gel
X 0.5 for heterozygous insertion
Mass of transgene DNA that is single copy
2740
3 X 109
X
5 X 10-6 g
=
4.567 X
10-12g
Haploid mouse genome, bp
=
4.567 pg
So a single copy standard will need 4.567 pg of transgene DNA added
to 10 ug genomic tail DNA
To use plasmid DNA that is only partly made up of the transgene
target sequence, determine the fraction that is the insert
sequence. e.g.:
2740 bp 5740 bp
=
0.477
Size of entire plasmid
Fraction of the plasmid that is the target sequence band
