Team:HokkaidoU Japan/Projects/asB0034/Method
From 2014.igem.org
RNA constructs
Anti-sense RBS fragments were synthesized based on BioBrick by primer annealing. The sides of antisense fragment have scar and restriction enzymes XhoI, NcoI. To be suitable BioBrick, we add scar sequence to anti-sense RNA. We finished synthesizing anti-sense RNA, we ligated anti-sense RNA with H-stem construction by restriction enzyme XhoI, NcoI.
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