Calculation of Copy Standards for PCR

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Calculation of Copy Standards for PCR

Copy Standard Calculation for PCR

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 0.1 micrograms of DNA to spike
  • Transgenic founder mice are heterozygous

Size of PCR band to be amplified from transgene, 825 bp in this example

0.1 ug genomic DNA per PCR reaction

X 0.5 for heterozygous insertion= 0.05

Mass of transgene DNA that is single copy

825 bp
3 X 109

X

0.05 X 10-6 g

=

13.75 X 10-15 g

Haploid mouse genome, bp

=

0.01375 pg

So a single copy standard will need 0.01375 pg of transgene DNA added to 0.1 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.:

825 bp
9340 bp

=

0.0883

Size of entire plasmid

Fraction of the plasmid that is the target sequence band

0.01375 pg
0.0883

=

0.1557 pg = 1X

 

see example of PCR using copy standards

0.7786 pg = 5X
1.5572 pg = 10X
7.7860 pg = 50X