10µg genomic DNA is digested and loaded onto one lane of a gel.
Each cell has 2pg DNA
First calculate how many cells are needed to generate the amount of DNA loaded onto each lane
Next calculate the weight of one mole plasmid (pGB-20 done here)
Next calculate the weight of one molecule plasmid
Then calculate how many grams of plasmid would be in each lane if every cell had exactly one plasmid in it
Therefore if every CHO cell had one copy of plasmid, and 10µg genomic CHO cell DNA were loaded into lane, then each lane would contain 38pg of plasmid DNA.
To get a good estimate of copy number in the cells, a series of copy number standards is run on the gel. For example:
1 copy = 38pg plasmid
10 copies = 380pg plasmid
50 copies = 1.9ng plasmid
100 copies = 3.8ng plasmid
250 copies = 9.5ng plasmid
500 copies = 19 ng plasmid
To get comparable signal strength and similar electrophoresis patterns, each lane of a gel should contain the same amount of total DNA in the same buffer. Therefore, each of the copy number standards is mixed with non-specific DNA, such as salmon sperm DNA, corresponding to the amount of genomic CHO cell DNA being assayed (10µg in this example).
Each DNA sample is then digested an equal length of time, in an equal volume, with the same restriction enzyme (Bgl I or Bgl II in the case of pGB-20). To get complete digestion, digest once for four hours, add more restriction enzyme, and allow digestion to continue overnight.
Since the volume is now too large to be loaded onto a single well, precipitate the DNA with 1/10 volume 3M sodium acetate and 2 volumes ethanol, centrifuge, wash pellet, resuspend in appropriate volume of 1x loading buffer, and run Southern blot.
The high copy number standards will generate a strong signal, so I always leave room between the standards and the sample being assayed when loading the gel.