Data Reduction

Data Reduction

After scanning，the plasmid and genomic DNA peak area values were entered into the plasmid copy number calculation form（we find a Microsoft Excel table format to be the most convenient）.See Table 1 for a representative example.The genomic DNA peak area for undiluted sample should be＞20.If it is＜20，the amount of DNA is not enough for quantitation，and the assay should be repeated with a double amount of cells.Adjusted genomic and plasmid DNA peak areas（@dil 1 line）were calculated by multiplying the peak area values by the corresponding dilution factors for all dilutions.To determine the linear range for the plasmid and genomic DNA peak areas at twofold sample dilutions，the percentage difference（%dif）between the adjusted value for each dilution and the adjusted value for each previous dilution（e.g.，16→8，32→16，64→32）was calculated using the formula：

The linear range was considered to be the range of values that differed by less than 20%.

The adjusted values in the linear range to be used for copy number calculation were entered into the Data Used line.For genomic DNA，the selected Data Used values in the linear range were limited only to the values originating from unadjusted peak areas in the range between 1.00 to 6.00.For plasmid DNA，the selected Data Used values in the linear range were limited only to the values originating from unadjusted peak areas in the range between 0.10 and 1.00.If only one peak value for plasmid DNA was detected，the number was used as it was.If no linear range could be determined，or more than one linear range was found，the solution in each particular case would be as follows：① If no linear range for pDNA1 and pDNA2（～7 and～6 kb pDNA）bands was determined，all the adjusted values for unadjusted peak areas in the range between 0.10 and 1.00 were taken into calculation.② The absence of linear range for genomic gDNA or～4 kb plasmid pDNA3 bands would indicate that an operational error must have occurred either during sample dilution or loading into the gel.Since the gDNA and pDNA3 peak area values are the most critical for the final result，in the latter case the sample must have been rediluted，and the assay repeated starting from the agarose gel electrophoresis step.③ If the linear range of gDNA could not be found only because the sample DNA was too high，then further dilution（＞256-fold）was the solution.④ If more than one linear range was found for pDNA bands，then the values in all the linear ranges，as well as the values between them，were entered into Data Used line.⑤ If there was only one Data Used point in the linear range for genomic DNA，then%dif with respect to the previous value must have been between-10%and+20%.If it was not between-10%and+20%，the sample DNA was rediluted，and the assay was repeated starting from the agarose gel electrophoresis step.

The values from the Data Used lines were averaged.The average value for genomic DNA was entered into the Total gDNA line.The sum of average values for plasmid DNA bands was entered into the Total pDNA line.Finally，the plasmid copy number was calculated using the following formula：Plasmid copy number（PCN）per genome

For example，

IL-2 plasmid copy number（PCN）per genome