Difference between revisions of "Part:BBa K4344070"

Revision as of 15:42, 12 October 2022

tac-LacO-SD-p19-rev.

Part of our primer set used for insertion of tac-LacO-shine Dalgarno element together with: BBa_K4344069 (tac-LacO-SD-p19-fwd.), BBa_K4344070 (tac-LacO-SD-p19-rev.), BBa_K4344071 (tac-Tac Extension-rev.)

Usage and Biology

We inserted a 7 bp long fragment by primer extension PCR between the tac promoter and the p19 starting codon using the primers sets pUC19-pBR322-ori-fwd./tac-tac Extension-rev. and p19-tac Extension-fwd/p19-HindIII-rev in two different PCR reactions as described earlier with pUC19-p19-UL19 siRNA serving as template. Success of PCR was validated on 1.2 % TAE-Agarose gel stained with ethidium bromide. All successful PCR products were pooled and recovered with the QIAGEN PCR Clean Up Kit and concentration was measured with Nanodrop 2000. Both products were used as a template at equimolar ratios for ligation of both fragments by PCR using the primers pUC19-pBR322-ori-fwd. and p19-HindIII-rev. Results were validated on 1.2 % TAE-Agarose gel. The PCR product with the correct size (502 bp) was extracted from the gel and recovered with the Qiagen Gel Extraction Kit.

After the successful cloning of pUC19-p19-siRNA UL19, we extend the insertion by a lac operator sequence and a Shine-Dalgarno sequence using the same principle. The lastly obtained plasmids were analysed by restriction digest with ClaI and HindIII on a 1.2 % agarose gel stained with ethidium bromide. We compared the obtained fragment sizes to the theoretical sizes acquired by digest in NEB Cutter v3.

Afterwards the Plasmid was sequenced with p19F and AmpR to confirm correct insertion of both inserts and conservation of loop structure as well as integrity of p19 gene. Furthermore the plasmid was sequenced with pUC19-pBR322ori-fwd to confirm complete presence of tac-lacO-Shine Dalgarno structural element.

Proposed structure of the complete pUC19-p19-siRNA UL19 (tac-LacO-SD).