Figure 2. Example of Second Round gp120 PCR plate agarose gel image.

The two samples used on the top row of the gel are undiluted genomic DNA from spleen tissue of two patients. Both samples have a total number of positive wells that equals less than 30%, indicating that the positive wells are most likely the result of nested PCR amplification of gp120 from a single integrated proviral genome in the DNA present in that well. The bottom row (HC09SPd1 DIL_1) provides an example of serial dilution testing to assess the correct SGS dilution. Four dilutions are tested here, and while all four dilutions are high enough to generate the amplification of a single integrated provirus in a positive well, all four are too high of a dilution to get many positive reactions resulting in wasted reagents. The ideal situation would be to find a dilution where 20-30% of the wells are positive, so lower dilutions must be tested to find an optimal dilution. The negative control, while not labeled on the gel, is in well A1, and the positive control (labeled POS) is in well H12. The negative control has 1 µl of the water used for dilution of the DNA, and the positive control is diluted genomic DNA from a patient who was not on cART that was PCR positive in previous experiments.