Journal: BMC Genomics
Article Title: Knockout of an outer membrane protein operon of Anaplasma marginale by transposon mutagenesis
Figure Lengend Snippet: Transcriptional analysis of the effect of the insertion of the Himar1 transposon within the omp10 gene by RT-PCR. A . Binding sites of primers (AB1553-AB1554), (AB1555-AB1556), (AB1591-AB1592), (AB1559-AB1560), and (AB1561-AB1562), designed to amplify transcripts on omp6, 7, 8, 9 and 10 , respectively, in wild-type (WT) and o mp10::himar1 mutant. Complementary DNA from WT and o mp10::himar1 mutant grown in ISE6 tick cells was used for PCR amplification for o mp6 through 10 with specific primers to evaluate gene expression. B . Agarose gel analysis of PCR products for omp6 through 10 in omp10::himar1 mutant (lanes 2, 8, 14, 20, and 26). PCR products for o mp6 through 10 in WT (lanes 5, 11, 17, 23, and 29). Genomic DNA was used as positive control (lanes 4, 7, 10, 13, 16, 19, 22, 25, 28, and 31). Complementary DNA from reactions without reverse transcriptase were used as negative controls (lanes, 3, 6, 9, 12, 15, 18, 21, 24, 27, and 30). 100 bp/1Kb DNA ladder lane 1). 16S rRNA (AB1572-AB1573) was used as an internal control to ensure integrity of cDNA (lanes 32–37).
Article Snippet: Based on this, the strategy that we used to map the Himar1 insertion site involved alignment of the sequencing reads obtained by Roche/454 and Illumina methods to two reference sequences, the A. marginale str.
Techniques: Reverse Transcription Polymerase Chain Reaction, Binding Assay, Mutagenesis, Polymerase Chain Reaction, Amplification, Expressing, Agarose Gel Electrophoresis, Positive Control