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Team:HokkaidoU Japan/Projects/asB0034/Method
From 2014.igem.org
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<div>Fig1. How to make anti-sense B0034 by primer annealing</div> | <div>Fig1. How to make anti-sense B0034 by primer annealing</div> | ||
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<div>Fig2. Using restriction enzyme, XhoI, NcoI, we made stem_anti-sense conplex. </div> | <div>Fig2. Using restriction enzyme, XhoI, NcoI, we made stem_anti-sense conplex. </div> | ||
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<div>Fig3. Blue; antisense B0034, B0032 Red; scar sequence Green; NcoI site Purple; XhoI site</div> | <div>Fig3. Blue; antisense B0034, B0032 Red; scar sequence Green; NcoI site Purple; XhoI site</div> | ||
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<div>Fig4. Our parts</div> | <div>Fig4. Our parts</div> | ||
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<div>Fig4. Anti-sense B0034 is induced by IPTG</div> | <div>Fig4. Anti-sense B0034 is induced by IPTG</div> | ||
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Revision as of 11:36, 14 October 2014
RNA constructs
Anti-sense RBS fragment was synthesized by primer annealing. Based on BioBrick standard, anti-senes RBS was flanked with scar sequences. Moreover, the ends of anti-sense fragment have restriction enzymes recognition sites, NcoI and XhoI. After finishing synthesizing anti-sense RNA, we ligated anti-sense RNA with H-stem construction by NcoI and XhoI.
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
Fig4. Our parts
How to assay
We selected mRFP for target gene. We used fluorophotometer to measure how anti-sense worked. The colonies transformed by anti-sense RNA and target gene was used for assay.
- To cultivate the colony in 4 mL LB culture for about 20 hours
- To control turbidity up to 0.1 at OD600
- To cultivate the colony in 2 mL M9ZB culture for 9 hours (IPTG induces antisense RNA, addition 20 uL)
- To measure fluorescence after 9 hour
Fig4. Anti-sense B0034 is induced by IPTG
