How to synthesize anti-sense constructs

Anti-sense fragments were synthesized based on BioBrick by PCR. Forward primers are common (XhoI-Ptet (-10)). Each reverse primers are different (as90 NcoI, as120 NcoI) (Fig. 1). As90 is the anti-sense that covers 90 bp of mRNA, and as 120 is the anti-sense that covers 120 bp of mRNA (complement RBS and a part of mRFP sequence.) Because of these, we got various length anti-senses. The sides of antisense fragment have restriction enzymes XhoI, NcoI sites.

Fig. 1 Synthesizing anti-sense by PCR

Fig. 2 Ligate the anti-sense fragment with H-stem vector

After we finished synthesizing pre anti-sense fragments, we cut them and H-stem vector (our anti-sense expression vector) by XhoI and NcoI. Finally, we ligated them. The anti-sense constructs are complete because pre anti-sense fragments are ligated reversely. Then, we measured their repression efficiencies. Therefore, we got anti-sense fragment, as90 and as120. As the same way, we made as30, as60 in H-Stem System and Anti-sense B0034 examination. We performed repression examination by using their 4 anti-sense constructs.

How to assay

We selected mRFP for the target gene. We used fluorophotometer to measure how anti-sense worked. The colonies transformed by anti-sense constructs and target gene was used for assay.

1. To cultivate the colony in 4 mL LB culture for about 20 hours
2. To control turbidity up to 0.1 at OD600
3. To cultivate the colony in 2 mL M9ZB culture for 9 hours (100mM IPTG induces antisense constructs, addition 20 uL)
4. To measure fluorescence after 9 hour