Team:HokkaidoU Japan/Projects/asB0034/Method

From 2014.igem.org

(Difference between revisions)
Line 99: Line 99:
<div class="fig fig400 para">
<div class="fig fig400 para">
-
<img src="https://static.igem.org/mediawiki/2014/3/38/HokkaidoU_project_antisenseB0034_overview02_800.png">
+
<img src="https://static.igem.org/mediawiki/2014/d/d1/HokkaidoU_project_antisenseB0034_method02_400.png">
<div>Fig1. How to make anti-sense B0034 by primer annealing</div>
<div>Fig1. How to make anti-sense B0034 by primer annealing</div>
</div>
</div>
<div class="fig fig400 para"><p>
<div class="fig fig400 para"><p>
-
<img src="https://static.igem.org/mediawiki/2014/3/38/HokkaidoU_project_antisenseB0034_overview03_800.png">
+
<img src="https://static.igem.org/mediawiki/2014/b/b9/HokkaidoU_project_antisenseB0034_method03_400.png">
<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>
</div>
</div>
<div class="fig fig800">
<div class="fig fig800">
-
<img src="https://static.igem.org/mediawiki/2014/3/38/HokkaidoU_project_antisenseB0034_overview5_800.png">
+
<img src="https://static.igem.org/mediawiki/2014/0/05/HokkaidoU_antisenseB0034_overview11.png">
<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>
</div>
</div>
<div class="fig fig800">
<div class="fig fig800">
-
<img src="https://static.igem.org/mediawiki/2014/3/38/HokkaidoU_project_antisenseB0034_overview6_800.png">
+
<img src="https://static.igem.org/mediawiki/2014/3/37/HokkaidoU_project_antisenseB0034_method06_400.png">
<div>Fig4. Our parts</div>
<div>Fig4. Our parts</div>
</div>
</div>
Line 130: Line 130:
<div class="fig fig800">
<div class="fig fig800">
-
<img src="https://static.igem.org/mediawiki/2014/3/38/HokkaidoU_project_antisenseB0034_overview04_800.png">
+
<img src="https://static.igem.org/mediawiki/2014/5/59/HokkaidoU_project_antisenseB0034_method04_800.png">
<div>Fig4. Anti-sense B0034 is induced by IPTG</div>
<div>Fig4. Anti-sense B0034 is induced by IPTG</div>
</div>
</div>

Revision as of 11:45, 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.

  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