Today we ran a gel of out PCR products too analyze the results. We also extracted DNA from the valencia leaves again since we ran out of the first batch. Bands produced by the amplified product were considerably brighter, suggesting that the increased elongation time was beneficial. Once again, the 42º lane produced maximal results. Unfortunately, it appears that the amplified product in that lane was smaller in size than our gene by a large margin (product ~ 800-1000bp, gene = 1600-1700bp). Furthermore, a particularly perplexing result of a large, approximately 3000bp fragment, appeared in lane B (anneal T = 59.2ºC). Though not ideal, a larger than gene fragment is a more favorable outcome than a smaller one because it suggests that introns are present within the amplified product. This is a predictable possibility for which we have prepared to proceed.
We must not draw undue conclusions from the lane B amplification. After all, it could just be a piece of turd or anything else for that matter. For this reason, we will be performing another PCR to get a better idea of what we see.
In addition, we will need to extract additional DNA from the pulverized leaf tissue because we are currently out.