Team:HokkaidoU Japan/Projects/asB0034/Method

From 2014.igem.org

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<h1>RNA constructs
<h1>RNA constructs
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<h4><p>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. </p>
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<h4><p>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. </p>
<div class="fig fig400 para">
<div class="fig fig400 para">
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<h1><p>How to assay</p>
<h1><p>How to assay</p>
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<h4><p>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.</p>
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<h4><p>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.</p>
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<ol>
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<p>(1)To cultivate the colony in 4 mL LB culture for about 20 hours</p>
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<li>To cultivate the colony in 4 mL LB culture for about 20 hours</li>
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<p>(2)To control turbidity up to 0.1 at OD600</p>
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<li>To control turbidity up to 0.1 at OD600</li>
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<p>(3)To cultivate the colony in 2 mL M9ZB culture for 9 hours (IPTG induces antisense RNA, addition 20 uL )</p>
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<li>To cultivate the colony in 2 mL M9ZB culture for 9 hours (IPTG induces antisense RNA, addition 20 uL)</li>
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<p>(4)To measure fluorescence after 9 hour </p>
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<li>To measure fluorescence after 9 hour</li>
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</ol>
<div class="fig fig800">
<div class="fig fig800">

Revision as of 11:20, 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

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