For the trial shown in Figure 2, 2.5 to 10 microliters of the eluted product was used in a 50 microliters PCR reaction. As seen in Figure 2, reamplification was accomplished for both products only when 2.5 microliters of template was used, while larger amounts of eluted sample consistently failed to re-amplify. Differential expression of the two sequences reported here was confirmed by Northern analysis. An mRNA Northern blot probed with the radiolabelled D9U10 sequence is shown in Figure 3. Only cells grown in the presence of nitric oxide trans-gene expression express a 4 kilobase mRNA identified using this probe.
Differential display is a challenging technique to master.
The reamplification of DNA sequences eluted from denaturing acrylamide gels must be consistent and reliable. When attempting to reamplify these valuable sequences, it should be kept in mind that including more of the eluted DNA sample in the reamplification PCR mixture is not necessarily better. Autoradiograph of a differential display gel. Boxes highlight two PCR products found to be differentially expressed. Reamplification of the differentially expressed DNA fragments.