Figure 2

a. An example of the PCR read out after limiting dilution. Cells were seeded at an average of 125 cells per well on a 96 well plate (see Table 1 on the derivation of the number of cells per well). Thirty samples, each representing on average 125 cells, were expanded to 10 ml cultures. PCR was performed to detect the LTR using primers NI2F and NI2R (see Methods). Ten samples were positive (asterisked). To control for the quality of DNA extraction, puromycin selected cells (lane S) were included as a positive extraction and PCR control, while untransduced Jurkat cells were used as the corresponding negative control (J). Plasmid HVP (P) and water (W) were used as positive and negative reaction controls respectively. The proportion of cells containing a provirus is 10/(30 × 125) = 0.267%. A further refinement of this estimate is to take into account that each positive signal could arise from more than one cell containing the provirus and their distribution into each well is essentially random. In this case the average number of cells containing the provirus in each well would be -ln(20/30) = 0.4054. Since each well represented 125 cells, the proportion of successfully transduced cell was 0.4054/125 = 0.324%. b. The concentration of plasmid HVP was determined by spectrophotometry, which was then diluted and subjected to PCR. The amount of plasmid input from lanes 1 to 8 are: 10 pg, 1 pg, 100 fg, 50 fg, 33 fg, 25 fg, 13 fg, and 10 fg respectively. Lane 9 is a negative control, amplifying water only. The limit of detection was at lane 6 with the DNA input of 25 fg, corresponding to an input of approximately 2300 copies of plasmid.