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Team:HokkaidoU Japan/Notebook/Length/as90 120
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<div class="ldd_submenu" style="left:-415px; width:970px;"> | <div class="ldd_submenu" style="left:-415px; width:970px;"> | ||
<ul style="margin-left:20px;"> | <ul style="margin-left:20px;"> | ||
| - | <li class="ldd_heading"><a href="https://2014.igem.org/Team:HokkaidoU_Japan/Projects/Overview/ | + | <li class="ldd_heading"><a href="https://2014.igem.org/Team:HokkaidoU_Japan/Projects/Overview/Background">Overview</a></li> |
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<li class="ldd_contents"><a href="https://2014.igem.org/Team:HokkaidoU_Japan/Projects/Overview/Background">Background</a></li> | <li class="ldd_contents"><a href="https://2014.igem.org/Team:HokkaidoU_Japan/Projects/Overview/Background">Background</a></li> | ||
<li class="ldd_contents"><a href="https://2014.igem.org/Team:HokkaidoU_Japan/Projects/Overview/Achievements">Achievements</a></li> | <li class="ldd_contents"><a href="https://2014.igem.org/Team:HokkaidoU_Japan/Projects/Overview/Achievements">Achievements</a></li> | ||
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<ul> | <ul> | ||
<li class="ldd_heading"><a href="https://2014.igem.org/Team:HokkaidoU_Japan/Projects/Length">Length Variation</a></li> | <li class="ldd_heading"><a href="https://2014.igem.org/Team:HokkaidoU_Japan/Projects/Length">Length Variation</a></li> | ||
| - | <li class="ldd_contents"><a href="">Overview</a></li> | + | <li class="ldd_contents"><a href="https://2014.igem.org/Team:HokkaidoU_Japan/Projects/Length">Overview</a></li> |
<li class="ldd_contents"><a href="https://2014.igem.org/Team:HokkaidoU_Japan/Projects/Length#Method">Method</a></li> | <li class="ldd_contents"><a href="https://2014.igem.org/Team:HokkaidoU_Japan/Projects/Length#Method">Method</a></li> | ||
<li class="ldd_contents"><a href="https://2014.igem.org/Team:HokkaidoU_Japan/Projects/Length#Results">Results</a></li> | <li class="ldd_contents"><a href="https://2014.igem.org/Team:HokkaidoU_Japan/Projects/Length#Results">Results</a></li> | ||
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</ul> | </ul> | ||
<ul> | <ul> | ||
| - | <li class="ldd_heading"><a href="https://2014.igem.org/Team:HokkaidoU_Japan/Outreach/Survey">High | + | <li class="ldd_heading"><a href="https://2014.igem.org/Team:HokkaidoU_Japan/Outreach/Survey">High School</a></li> |
<li class="ldd_contents"><a href="https://2014.igem.org/Team:HokkaidoU_Japan/Outreach/Survey#Education">Education</a></li> | <li class="ldd_contents"><a href="https://2014.igem.org/Team:HokkaidoU_Japan/Outreach/Survey#Education">Education</a></li> | ||
<li class="ldd_contents"><a href="https://2014.igem.org/Team:HokkaidoU_Japan/Outreach/Survey#Survey">Survey</a></li> | <li class="ldd_contents"><a href="https://2014.igem.org/Team:HokkaidoU_Japan/Outreach/Survey#Survey">Survey</a></li> | ||
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<li class="ldd_heading"><a href="https://2014.igem.org/Team:HokkaidoU_Japan/Outreach/Discussion">Discussion</a></li> | <li class="ldd_heading"><a href="https://2014.igem.org/Team:HokkaidoU_Japan/Outreach/Discussion">Discussion</a></li> | ||
<li class="ldd_contents"><a href="https://2014.igem.org/Team:HokkaidoU_Japan/Outreach/Discussion#Background">Background</a></li> | <li class="ldd_contents"><a href="https://2014.igem.org/Team:HokkaidoU_Japan/Outreach/Discussion#Background">Background</a></li> | ||
| - | <li class="ldd_contents"><a href="https://2014.igem.org/Team:HokkaidoU_Japan/Outreach/Discussion# | + | <li class="ldd_contents"><a href="https://2014.igem.org/Team:HokkaidoU_Japan/Outreach/Discussion#Evaluation">Evaluation</a></li> |
</ul> | </ul> | ||
</div> | </div> | ||
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</ul> | </ul> | ||
<div class="step-recipe"> | <div class="step-recipe"> | ||
| - | R0040-B0034-E1010-B0015 | + | R0040-B0034-E1010-B0015 |
| + | </div> | ||
| + | <div class="step-recipe"> | ||
| + | as_RBS_XhoI_F Tm:68.2℃ & 90bp_asmRFP_Nco_R Tm:79.9℃ | ||
| + | </div> | ||
| + | <div class="step-recipe"> | ||
| + | as_RBS_XhoI_F Tm:68.2℃ & 120bp_asmRFP_Nco_R Tm:66.9℃ | ||
| + | |||
</div> | </div> | ||
</div> | </div> | ||
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</ul> | </ul> | ||
<div class="step-recipe"> | <div class="step-recipe"> | ||
| - | Cut | + | Cut as90(on H-stem) with NcoI,XhoI(using 10×Cut Smart) |
| + | </div> | ||
| + | <div class="step-recipe"> | ||
| + | Cut as120(on H-stem) with NcoI,XhoI(using 10×Cut Smart) | ||
</div> | </div> | ||
</div> | </div> | ||
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<div class="step-tile"> | <div class="step-tile"> | ||
<h2 class="step-title"> | <h2 class="step-title"> | ||
| - | + | Dephosphorylation | |
</h2> | </h2> | ||
</div> | </div> | ||
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</ul> | </ul> | ||
<div class="step-recipe"> | <div class="step-recipe"> | ||
| - | H-stem | + | Antactic phosphorylate H-stem (using 10×Antarctic phosphatase buffer) 37℃ 35min 65℃ 10min |
| + | |||
</div> | </div> | ||
</div> | </div> | ||
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</ul> | </ul> | ||
<div class="step-recipe"> | <div class="step-recipe"> | ||
| - | Ligate as90, as120 with H-stem | + | Ligate as90, as120 with H-stem(on pSB1A3) |
| + | |||
</div> | </div> | ||
</div> | </div> | ||
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</ul> | </ul> | ||
<div class="step-recipe"> | <div class="step-recipe"> | ||
| - | as90, as120 on | + | as90, as120(on pSB1A3) |
| + | </div> | ||
| + | <div class="step-recipe"> | ||
| + | 5µL DNA to DH5α Turbo | ||
</div> | </div> | ||
</div> | </div> | ||
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</ul> | </ul> | ||
<div class="step-recipe"> | <div class="step-recipe"> | ||
| - | as90 on | + | as90 (on pSB1A3) XhoI_RBS_ Tm:68.2℃ & as90_NcoI_ Tm:79.9℃ |
</div> | </div> | ||
| + | <div class="step-recipe"> | ||
| + | as120(on pSB1A3) XhoI_RBS_ Tm:68.2℃ & as120_NcoI_ Tm:66.9℃ | ||
| + | </div> | ||
| + | <div class="step-recipe"> | ||
| + | Kapa-Taq | ||
| + | </div> | ||
| + | |||
</div> | </div> | ||
</div> | </div> | ||
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</ul> | </ul> | ||
<div class="step-recipe"> | <div class="step-recipe"> | ||
| - | + | R0010-K1524104-B0015(on pSB1A3) | |
| + | </div> | ||
| + | <div class="step-recipe"> | ||
| + | R0010-K1524105-B0015(on pSB1A3) | ||
| + | |||
</div> | </div> | ||
</div> | </div> | ||
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</ul> | </ul> | ||
<div class="step-recipe"> | <div class="step-recipe"> | ||
| - | + | R0010-K1524104-B0015(on pSB1A3) | |
| + | </div> | ||
| + | <div class="step-recipe"> | ||
| + | R0010-K1524105-B0015(on pSB1A3) | ||
| + | |||
</div> | </div> | ||
</div> | </div> | ||
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</ul> | </ul> | ||
<div class="step-recipe"> | <div class="step-recipe"> | ||
| - | + | R0010-K1524104-B0015(on pSB1A3) | |
| + | </div> | ||
| + | <div class="step-recipe"> | ||
| + | R0010-K1524105-B0015(on pSB1A3) | ||
| + | |||
</div> | </div> | ||
</div> | </div> | ||
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<div id="footer-content"> | <div id="footer-content"> | ||
<div id="footer-logo"> | <div id="footer-logo"> | ||
| - | <a href="http://igemhokkaidou.com | + | <a href="http://igemhokkaidou.wordpress.com"><img style="height:150px;position:relative;" src="https://static.igem.org/mediawiki/2014/3/39/HokkaidoU_logo_transparent.png"></a> |
</div> | </div> | ||
<div id="footer-twitter"> | <div id="footer-twitter"> | ||
Latest revision as of 15:32, 9 September 2015
Notebook
Lab Documents
Anti-sense 90, 120 Construction
-
Start
-
PCR & PCR purification
R0040-B0034-E1010-B0015as_RBS_XhoI_F Tm:68.2℃ & 90bp_asmRFP_Nco_R Tm:79.9℃as_RBS_XhoI_F Tm:68.2℃ & 120bp_asmRFP_Nco_R Tm:66.9℃ -
Digestion
Cut as90(on H-stem) with NcoI,XhoI(using 10×Cut Smart)Cut as120(on H-stem) with NcoI,XhoI(using 10×Cut Smart) -
Ethanol precipitation
as90, as120 -
Gel Extraction
H-stem -
Dephosphorylation
Antactic phosphorylate H-stem (using 10×Antarctic phosphatase buffer) 37℃ 35min 65℃ 10min -
Ligation
Ligate as90, as120 with H-stem(on pSB1A3) -
Transformation
as90, as120(on pSB1A3)5µL DNA to DH5α Turbo -
Colony PCR
as90 (on pSB1A3) XhoI_RBS_ Tm:68.2℃ & as90_NcoI_ Tm:79.9℃as120(on pSB1A3) XhoI_RBS_ Tm:68.2℃ & as120_NcoI_ Tm:66.9℃Kapa-Taq -
Liquid Culture
R0010-K1524104-B0015(on pSB1A3)R0010-K1524105-B0015(on pSB1A3) -
Mini-prep
R0010-K1524104-B0015(on pSB1A3)R0010-K1524105-B0015(on pSB1A3) -
Dye terminator
R0010-K1524104-B0015(on pSB1A3)R0010-K1524105-B0015(on pSB1A3) -
Complete!
