How to synthesize anti-sense RNA

Anti-sense 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 anti-sense B0034 by primer annealing

Fig2. Using restriction enzyme, XhoI, NcoI, we made stem_anti-sense 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 anti-sense worked. The colonies transformed anti-sense 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. Anti-sense B0034 is induced by IPTG