a&w restaurant: How does a southern blot provide information about gene copy number? So my book says that chromosomal DNA is digested with restriction enzymes first. Does it depend on the number of fragments in the different lanes(corresponding to different restriction enzymes) or do we assess based on the intensity compared to some baseline value?|||Copy number is calculated by comparing the signal intensity of your unknown copy number to a known copy number series. Your known is a standard that is blotted using picograms equal to 1 copy, 5 copies, 10 copies, etc (hopefully of the cloned cDNA of your gene) so the fragment length is the same. You know the normal copy number of a gene is two alleles in a diploid species but copy number can be higher in polyploid plants or it can be higher in transgenic chimeric constructs with multiple inserts. Here is an example...just substitute your plant's numbers for the human haploid genome size of 3 X 10^9 bp: Assumption: the Haploid content of the genome is 3 X 10^9 bp Assumption: you have 2 micrograms of gDNA available Assumption: the gene of interest is 5,480 bp and you have a cDNA cloned You want to determine the copy number of your gene to assay by Southern and you need a standard to calibrate band intensities. Since transgenic founder mice are hemizygous, only one chromosome has the inserted gene(s), use the haploid value to find 1 copy's mass: Find the mass of the transgene/1 ug of gDNA = N bp transgene DNA/3 X 10^9 bp gDNA Now, for a 5,480 bp gene/transgene..re-arrange the equation to: mass of gene DNA = 2 x <(5,480 bp cloned DNA) X (1 碌g gDNA)/ 3 X 10^9 bp gDNA> mass of transgene cDNA for a standard = 3.66 picograms Thus, to prepare a 1 copy standard: use 3.66 pg of gene cDNA and compare signal intensity to 2 microgram of gDNA 0.1 copy 0.366 pg 1 copy 3.66 pg 10 copy 36.6 pg 50 copy 183 pg 100 copy 366 pg The 0.1 copy should be a control for the standard by being to little to detect and give a relative zero point.|||the size of the fragment will be larger if there is two copies compared to one copy. Consider a line - and it being bold, bold would be to copies and not bold would be one. Sorry not sure how else to explain this one!