Difference between revisions of "Part:BBa K4344059"

Latest revision as of 15:04, 12 October 2022

SalI-HSV-UL19-6xHis-rev.

Part of our primer set used for amplification of HSV-UL19 (Part: K4344020) with: BBa_K4344033 (NotI-HSV-UL19-6xHis-fwd.), BBa_K4344055 (SacI-HSV-UL19-6xHis-fwd.), BBa_K4344059 (SalI-HSV-UL19-6xHis-rev.), BBa_K4344075 (XhoI-HSV-UL19-6xHis-rev.)

Usage and Biology

Huang et al. recommend that the siRNA target area’s size should range between 250 and 500 bp (Huang et al., 2013). We selected a 249 bp area for UL19 (Sequence: 2527 – 2775; Fragment: 82 - 330). The siRNA target area includes the previously described structural motif. For EGFP knockdown a previously published siRNA sequence (sense: 5’-GCAAGCUGACCCUGAAGUUCAUTT-3’; (Metwally, A. A., Blagbrough, I. S., & Mantell, J. M., 2012) was chosen. The analysis of our target areas with siRNA Design Tool from IDT revealed 13 potential siRNA candidates for UL19. 2 of these 13 candidates were indicated as cross-reacting with human gene transcripts.

siRNA target areas were amplified with SacI/XhoI or SalI/NotI sequence extension by PCR. We obtained amplicons with a size ranging from 200 to 300 bp which was congruent with the expected sizes of 266 bp for the SacI/XhoI Primer set and 268 bp for the NotI/SalI primer set. After performing a two-step cloning and transformation in E. coli, we analysed the obtained plasmids by restriction digest with SacI and NotI to confirm the presence of the insert in both restriction cassettes. Results were visualised on a 1.2 % agarose gel stained with ethidium bromide. We compared the resulting fragment sizes to the theoretical sizes obtained by digest in NEB Cutter v3

1.2 % Analytical of UL19 siRNA target area PCR with primer sets SacI/XhoI and SalI/NotI