Selection markers Gene amplification

Figure 19 Sample gel of the results of a PCR. Lane 1 is a 100-bp molecular marker lanes 2-6 are samples. The presence of the top bands (the species-specific endogenous gene) demonstrates that the PCR amplification was successfiil. Lack of the middle band (the introduced effect gene) and the bottom band (the selectable marker gene) in lane 3 indicates that sample is negative for the effect gene. Presence of all three bands in the remaining lanes indicates the samples are positive for the effect gene...

Fig. 2. Examples of a differential display analysis. Total RNA was isolated from chicken embryos at two different embryonic stages. Five, 10, and 50 ng of total RNA were subjected to reverse transcription, and afterward to amplification for 35 cycles using the M13 reverse primer, under the conditions described in the text. The amplification products were then separated on a denaturing polyacrylamide gel (A). Several bands were selected (observe the holes punched to recover the products from the gel), and after selection, the gel was reexposed to verify recovery (not shown). The recovered material was subjected again to PCR under the conditions described in Subheading 3.2. As markers, 50 ng of cDNA were amplified independently as described in (A), to check again for the appearance of the products selected. These products are now cloned, sequenced, and the major product will be considered as the real differentially expressed gene. Until Northern blot analysis is performed, there is no previous clue about the authenticity of the products.

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