Figure 3 of
de Peyer, Mol Vis 1999;
Figure 3. Analysis of transgenic plants for the presence of MIP using PCR and RT PCR
A. MIP26 PCR product from plant genomic DNA. Plant genomic DNA was extracted using the Qiagen "DNAeasy" kit. Plant genomic DNA was used as the template in PCR, followed by agarose gel electrophoresis analysis. Lane 1, DNA size markers; lane 2, PCR without template; lane 3, wild-type plant DNA alone (these lanes show that PCR contamination precautions were adequate to prevent aerosol contamination and false positive bands); lane 4, lack of PCR products from wild-type plant DNA; lanes 5, 7 and 9, DNA from transgenic plants; lanes 6, 8, and 10, PCR products from DNA from transgenic plants. Lanes 6, 8, and 10 contained a band of about 800 bp in size, which was excised, sequenced, and confirmed as bovine MIP26.
B. MIP26 RT PCR product from plant mRNA. Plant mRNA was extracted using the Dynal Dynabeads mRNA DIRECT kit. Plant cDNA generated from plant mRNA after reverse transcription was both used as a templates in PCR, followed by agarose gel electrophoresis analysis. Reverse transcription was carried out using the Promega Access RT-PCR kit. Lanes 1 and 14, DNA size markers; lanes 2 and 3, RT-PCR with no template; lanes 4 and 5, positive control RT-PCR with known mRNA; lanes 6 and 7, wild-type plant mRNA (these lanes show that the primers employed were be specific enough to not inadvertently amplify native plant cDNAs); lanes 8, 9,10,11,12 and 13, mRNA from tansgenic plants. These reactions gave a band of about 800 bp in size, which was excised and sequenced, and confirmed as bovine MIP26. The positive control (lanes 4 and 5) was as supplied with the Promega Access RT-PCR kit (mRNA for the E. coli kanamycin resistance gene), giving rise to a 323 bp amplimer using the primers supplied with the kit.