Difference between revisions of "Part:BBa K4344075"

Revision as of 15:05, 12 October 2022

XhoI-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.)

Sequence and Features

Assembly Compatibility:

10
COMPATIBLE WITH RFC[10]
12
COMPATIBLE WITH RFC[12]
21
INCOMPATIBLE WITH RFC[21]
Illegal XhoI site found at 3
23
COMPATIBLE WITH RFC[23]
25
COMPATIBLE WITH RFC[25]
1000
COMPATIBLE WITH RFC[1000]

@@ Line 3: / Line 3: @@
 <partinfo>BBa_K4344075 short</partinfo>
-Placeholder
+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.)
 <!-- Add more about the biology of this part here
 ===Usage and Biology===
+Huang <i>et al.</i> recommend that the siRNA target area’s size should range between 250 and 500 bp <span class="scientific-src">(Huang <i>et al.</i>, 2013)</span>. We selected a 249 bp area for <i>UL19</i> (Sequence: 2527 – 2775; Fragment: 82 - 330). The siRNA target area includes the previously described structural motif. For <i>EGFP</i> knockdown a previously published siRNA sequence (sense: 5’-GCAAGCUGACCCUGAAGUUCAUTT-3’; <span class="scientific-src">(Metwally, A. A., Blagbrough, I. S., & Mantell, J. M., 2012)</span> was chosen.
+The analysis of our target areas with siRNA Design Tool from IDT revealed 13 potential siRNA candidates for <i>UL19</i>. 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 <i>E. coli</i>, 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
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