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). Because of these, we got various length anti-senses. The sides of antisense fragment have restriction enzymes XhoI, NcoI sites. We finished synthesizing anti-sense RNA, we ligated each anti-senses with H-stem vector by restriction enzyme XhoI, NcoI.

Fig. 1 Synthesizing anti-sense by PCR

Therefore, we got as90, 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