was digested with PstI (TaKaRa, Japan) to prevent the copy number from being underestimated (66).
Why does digestion improve the estimation of plasmid vs the chromosome? I would assume the chromosome is more tightly supercoiled and inaccessible unless digested right?
RSF1010 is maintained at a copy-number of about 10-12 per chromosome in E coli, P. aeruginosa and S. enterica sv.Typhimurium (Frey and Bagdasarian, 1989)
Copy numbers of the oriV region and gfp gene on the plasmid relative to a chromosomal gene were determined by qPCR. The chromosomal gene dapA was set up as the reference gene
Designed probes were modified at the 5′ end with the fluorophore FAM for the reference gene cysG and HEX for the plasmid marker oriT, and furthermore modified at the 3′ end with the quencher BHQ-1
plasmid copy number qPCR assay.
Why cysG: Siroheme synthase gene?