Calculation of Copy Standards

SOM Home > Research > Gene Targeting and Transgenic Facility > Protocols > Calculation of Copy Standards

Calculation of Copy Standards


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

 
 

4.567 pg
0.477

=

9.574 pg = 1X

 

See example of Southern blot with these controls.

 

47.872 pg = 5X

95.744 pg = 10X

478.72 pg = 50X