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Team:HokkaidoU Japan/Projects/asB0034/Method
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<img src="https://static.igem.org/mediawiki/2014/d/d1/HokkaidoU_project_antisenseB0034_method02_400.png"> | <img src="https://static.igem.org/mediawiki/2014/d/d1/HokkaidoU_project_antisenseB0034_method02_400.png"> | ||
| - | <div> | + | <div>Fig. 1 How to make anti-sense B0034 by primer annealing</div> |
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<img src="https://static.igem.org/mediawiki/2014/b/b9/HokkaidoU_project_antisenseB0034_method03_400.png"> | <img src="https://static.igem.org/mediawiki/2014/b/b9/HokkaidoU_project_antisenseB0034_method03_400.png"> | ||
| - | <div> | + | <div>Fig. 2 Using restriction enzyme, XhoI, NcoI, we made stem_anti-sense conplex. </div> |
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<img src="https://static.igem.org/mediawiki/2014/0/05/HokkaidoU_antisenseB0034_overview11.png"> | <img src="https://static.igem.org/mediawiki/2014/0/05/HokkaidoU_antisenseB0034_overview11.png"> | ||
| - | <div> | + | <div>Fig. 3 Blue; antisense B0034, B0032 Red; scar sequence Green; NcoI site Purple; XhoI site</div> |
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| + | <img src="https://static.igem.org/mediawiki/2014/c/cb/HokkaidoU_project_antisenseB0034_method01_400.png"> | ||
| + | <div>Fig. 4 B0034 & B0032 sequence </div> | ||
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<img src="https://static.igem.org/mediawiki/2014/3/37/HokkaidoU_project_antisenseB0034_method06_400.png"> | <img src="https://static.igem.org/mediawiki/2014/3/37/HokkaidoU_project_antisenseB0034_method06_400.png"> | ||
| - | <div> | + | <div>Fig. 5 Our parts</div> |
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<img src="https://static.igem.org/mediawiki/2014/5/59/HokkaidoU_project_antisenseB0034_method04_800.png"> | <img src="https://static.igem.org/mediawiki/2014/5/59/HokkaidoU_project_antisenseB0034_method04_800.png"> | ||
| - | <div> | + | <div>Fig. 6 Anti-sense B0034 is induced by IPTG</div> |
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Latest revision as of 11:58, 14 October 2014
How to synthesize anti-sense 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.
Fig. 1 How to make anti-sense B0034 by primer annealing
Fig. 2 Using restriction enzyme, XhoI, NcoI, we made stem_anti-sense conplex.
Fig. 3 Blue; antisense B0034, B0032 Red; scar sequence Green; NcoI site Purple; XhoI site
Fig. 4 B0034 & B0032 sequence
Fig. 5 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
Fig. 6 Anti-sense B0034 is induced by IPTG
