Agarose Gel Electrophoresis and Scanning of Negati...
Serial twofold dilutions of bacterial DNA sample（2，4，8，16，32，64，128，and 256-fold）were made with TE buffer.Ten microliters of undiluted and each diluted sample was mixed with 2 μl of 6×gel loading dye and loaded into the wells of 0.8%0.5×TBE agarose gel.Two hundred nanograms of λ DNA/HindIH digest was used as a molecular weight marker.The gel was run in 0.5×TBE buffer using Bio-Rad WIDE MINI-SUB CELL at 100 V until the bromophenol blue reached approximately 2 cm from the bottom of the gel（about 1 h）.The gel was stained with ethidium bromide（0.5 μg/ml）for 20 min and destained in water for 10 min.The photographs of the ethidium bromide-stained gels were taken under UV light with Polaroid positive/neg ative film No.55（exposure time is 1 min at F 4.5 with a yellow filter）.The negatives were developed by immersing in 18%sodium sulfite solution.Four bands could be detected on the gel（see Figure 2）：genomic DNA（gDNA，～23 kb mobility）；plasmid form with～7 kb mobility（pDNA1，linear form）；plasmid form with～6 kb mobility（pDNA2，nicked circular form）；and plasmid form with～4 kb mobility（pDNA3，supercoiled form）.The negatives were scanned using the Personal Densitometer SI（Molecular Dynamics）.The baseline was selected using the All Valleys Method according to the Gel Scanner User's Guide，i.e.，by drawing a straight line between the lowest points of the left and right peak shoulders（see Figure 3）.It is important to remember that using an incorrect baseline may significantly affect the resulting peak area and，consequently，the estimated plasmid copy number.
Figure 2 Serial twofold dilutions of total bacterial DNA sample run in 0.8%agarose gel
（a）Ethidium bromide-stained gel：MW is molecular weight marker（λ DNA/HindⅢ digest）；（b）Gel negative(https://www.daowen.com)
Figure 3 Typical negative scan result representing one agarose gel lane：peak 1 is gDNA；peak 2 is pDNA1；peak 3 is pDNA2；peak 4 is pDNA3
Scanner and image analysis software that can be used in this plasmid copy number assay is not limited to the Personal Densitometer SI（Molecular Dynamics）.One may use，for example，those available from Bio-Rad or Alpha Innotech.In our experiments，scanning the same negative with three different scanners yielded the same plasmid copy number.With software other than Molecular Dynamics，however，some adjustments in the Data Used values range might be necessary，i.e.，the selected range might be different from 1.00 to 6.00 for genomic DNA，and 0.10 to 1.00 for plasmid DNA（see data reduction rules below）.
