How to synthesize antisense RNA

Antisense-RBS fragments were synthesized based on BioBrick by primer annealing. The sides of antisense fragment have scar and restriction enzymes XhoI, NcoI.

Fig1. How to make antisense B0034 by primer annealing

Fig2. Using restriction enzyme, XhoI, NcoI, we made stem-antisense conplex.

Fig3. Blue; antisense B0034, B0032 Red; scar sequence Green; NcoI site Purple; XhoI site

How to assay

we selected RFP and GFP for target genes. We used fluorophotometer to measure how antisense worked. The colonies transformed antisense RNA and target gene used to do 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 (IPTG induces antisense RNA, addition 20 uL )

(4)To measure fluorescence after 9 hour

Fig4. Antisense B0034 is induced by IPTG