Preparation of Copy Standards for
Southern Blot Copy Number
Determination
and PCR Genotyping Sensitivity
PCR
screens must be designed to detect transgene DNA at the single copy
level or 0.1 copy level..
Southern Blots analysis of transgenic mice need copy standards to
estimate copy
number.
Copy standards are prepared by mixing non-transgenic tail DNA with a
known
amount of transgene DNA is to produce transgene copy standards.
For PCR, these standards can be used to determine the sensitivy of the
PCR
assay. You must obtain single copy sensitivity in your PCR prior to
transgene
submission. When you test DNA from potentially transgenic founder mice,
run
your single copy PCR test sample to ensure that your PCR assay is
sensitive
enough to avoid false negatives.
Southern Blots are commonly used to determine transgene copy number and
the
number of integration sites in transgenic founder mice.
Download a pdf file illustrating Southern Blot
analysis
of transgenic founders.
Calculation of Copy
Number Standards
Assumption:
the Haploid content of a mammalian genome is 3 X 109 bp
Assumption: you have 2 micrograms of tail DNA available
Since the transgenic founder mice are hemizygous:
mass of transgene DNA
=
N bp transgene DNA
1 microgram genomic DNA 3 X 109
bp genomic DNA
Example: for a 5,480 bp transgene insert or plasmid
mass of transgene DNA
=
5,480 bp cloned DNA
or
1 micrograms genomic DNA 3 X 109
bp genomic DNA
mass of transgene DNA = (5,480 bp cloned DNA) X (1 µg genomic
DNA)
or
3 X 109 bp genomic DNA
mass of transgene DNA = 1.83 picograms per 1 ug genomic DNA
transgenic mice will be hemizygous for the transgene, not homozygous
1.83pg transgene should be added to 2 ug of genomic DNA
Thus, to prepare a 1 copy standard: add 1.83 pg of transgene
DNA to 2
microgram tail DNA
0.1 copy 0.183 pg
10
copy
18.3 pg
50
copy
91.5
pg
100
copy 183
pg
For use as a transgene PCR standard, use 200 ng of the spiked tail DNA as a substrate in a 25 ul PCR reaction as described: genotyping transgenic mice.
For use in Southern blot analysis, digest the tail DNA as you would for Southern analysis, and add the transgene insert DNA (not the entire plasmid) just before you load your gel. Remember to reserve one lane for genomic DNA only with no spike. For an example of copy standards in Southern blots, refer to Camper SA. 1987. Research applications of transgenic mice. Biotechniques 5, 638-650. Click here for more review articles.
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