Protocol for genotyping by PCR
In order for the LRTGT to guarantee the production of three founder mice for transgenic analysis, we require that the investigator provides proof of a genotyping protocol that can detect a single copy insertion by PCR analysis.
This requires identifying primers that would be unique to the transgene and not be able to amplify any endogenous genomic regions. For primer design, we recommend using the program Primers3. This program can be used through the following website address (http://frodo.wi.mit.edu/). Simply paste the sequence of your entire transgene into the space provided and hit the button “pick primers”. You will be given several options to choose from.
To ensure these primers work and are specific, we ask that you perform PCR on mouse genomic DNA that has been spiked with your transgene to reflect a single copy of DNA within the genome.
To determine the amount of plasmid to add to genomic DNA, use the following calculations:
Weight of the transgene = (#bp in the transgene) x (660 daltons/bp) x (1.67 x 10-24 g/dalton)
Weight of the genome = (5.0 x 109 bp) x (660 daltons/bp) x (1.67 x 10-24 g/dalton)
(diploid genome) = 5.4 x 10-12 g
How many copies of the genome are in 20 µg of DNA?
20 µg /5.4 x 10-12 g = 3.8 x 106 copies of the genome
Therefore, 20 µg of DNA = 3.8 x 106 copies of the genome and
1 µg of DNA = 1.9 x 105 copies of the genome
Example
For a transgene that is 9200 bp (not including vector sequence), the amount of plasmid used to spike 1 µg of genomic DNA to the equivalent of a single copy of the transgene.
(#bp in the transgene) x (660 daltons/bp) x (1.67 x 10-24 g/dalton)
= (9200 bp) x (660 daltons/bp) x (1.67 x 10-24 g/dalton)
= 6,072,000 daltons x (1.67 x 10-24 g/dalton)
= 1.01 x 10-17 g
For a single copy representation of this transgene in 1 g of genomic DNA
= (weight of the transgene) x (1.9 x 105)
= (1.01 x 10-17 g) x (1.9 x 105)
= 1.92 x 10-12 g
= 1.92 picograms
For PCR analysis, we typically perform PCR on 0.5 to 1.0 g of genomic DNA.
