Team:HokkaidoU Japan/Notebook/Pre experiment/140910 Ptet-B0032-mRFP-dT

From 2014.igem.org

(Difference between revisions)
 
(6 intermediate revisions not shown)
Line 5: Line 5:
{{Team:HokkaidoU_Japan/LargeDropDown/CSS}}
{{Team:HokkaidoU_Japan/LargeDropDown/CSS}}
<html>
<html>
 +
<link href='http://fonts.googleapis.com/css?family=Merriweather' rel='stylesheet' type='text/css'>
<div id="background">
<div id="background">
<!--begin header-->
<!--begin header-->
Line 81: Line 82:
</ul>
</ul>
<ul>
<ul>
-
<li class="ldd_heading"><a href="https://2014.igem.org/Team:HokkaidoU_Japan/Outreach/Survey">High-School</a></li>
+
<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>
Line 88: Line 89:
<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#Estimation">Evaluation</a></li>
+
<li class="ldd_contents"><a href="https://2014.igem.org/Team:HokkaidoU_Japan/Outreach/Discussion#Evaluation">Evaluation</a></li>
</ul>
</ul>
</div>
</div>
Line 113: Line 114:
<div class="wrapper">
<div class="wrapper">
   <div id="hokkaidou-contents">
   <div id="hokkaidou-contents">
-
   <h2 class="step-title">P<sub>tet</sub>-B0032-mRFP-dT</h2>
+
   <h2 class="step-title">Ptet-B0032-mRFP-dT</h2>
     <div class="workflow-container">
     <div class="workflow-container">
       <ul class="steps">
       <ul class="steps">
Line 137: Line 138:
      </ul>
      </ul>
      <div class="step-recipe">
      <div class="step-recipe">
-
P<sub>tet</sub>-B0034-mRFP-dT(6A1) SpeI_PstI_F& P<sub>tet</sub>_SpeI_R(vector), XbaI_B0032_mRFP_F& PstI_SpeI_R(insert), KOD Fx Neo
+
R0040-B0034-E1010-B0015(on pSB6A1) SpeI_PstI_F Tm:72.8&#8451;& Ptet_SpeI_R Tm:72.6&#8451; (vector)
 +
</div>
 +
<div class="step-recipe">
 +
XbaI_B0032_mRFP_F Tm:76.3&#8451; & PstI_SpeI_R Tm:74.6&#8451; (insert)
 +
</div>
 +
<div class="step-recipe">
 +
KOD Fx Neo
</div>
</div>
    </div>
    </div>
Line 158: Line 165:
      </ul>
      </ul>
      <div class="step-recipe">
      <div class="step-recipe">
-
digested PCR productions
+
PCR products
</div>
</div>
    </div>
    </div>
Line 179: Line 186:
      </ul>
      </ul>
      <div class="step-recipe">
      <div class="step-recipe">
-
Cut  vector with SpeI, PstI (using 10&times;H buffer) Cut  insert with XbaI, PstI (using 10&times;M buffer)
+
Cut  vector with SpeI, PstI (using 10&times;H buffer)  
 +
</div>
 +
<div class="step-recipe">
 +
Cut  insert with XbaI, PstI (using 10&times;M buffer)
</div>
</div>
    </div>
    </div>
Line 187: Line 197:
  <div class="step-tile">
  <div class="step-tile">
  <h2 class="step-title">
  <h2 class="step-title">
-
Dephophorylation
+
Dephosphorylation
</h2>
</h2>
  </div>
  </div>
Line 201: Line 211:
      <div class="step-recipe">
      <div class="step-recipe">
Antactic phosphorylate insert (using 10&times;Antarctic phosphatase buffer) 37&#8451; 35min 65&#8451; 10min
Antactic phosphorylate insert (using 10&times;Antarctic phosphatase buffer) 37&#8451; 35min 65&#8451; 10min
 +
</div>
</div>
    </div>
    </div>
Line 242: Line 253:
      </ul>
      </ul>
      <div class="step-recipe">
      <div class="step-recipe">
-
ligation products on ice  30min each of 5.0&micro;L DNA to DH5&alpha;  42&#8451;        1min on ice 2min
+
R0040-B0032-E1010-B0015(on pSB1A6)
 +
</div>
 +
<div class="step-recipe">
 +
5.0&micro;L DNA to DH5&alpha;   
 +
 
</div>
</div>
    </div>
    </div>
Line 263: Line 278:
      </ul>
      </ul>
      <div class="step-recipe">
      <div class="step-recipe">
-
P<sub>tet</sub>-B0034-mRFP-dT on pSB6A1 EcoI_XbaI_F Tm:70.9&#8451; rbs_SpeI_R Tm:59.2&#8451;
+
R0040-B0032-E1010-B0015(on pSB6A1) EcoI_XbaI_F Tm:70.9&#8451; & RBS_SpeI_R Tm:59.2&#8451;
 +
 
</div>
</div>
    </div>
    </div>
Line 284: Line 300:
      </ul>
      </ul>
      <div class="step-recipe">
      <div class="step-recipe">
-
P<sub>tet</sub>-B0034-mRFP-dT on pSB6A1
+
R0040-B0032-E1010-B0015(on pSB6A1)
</div>
</div>
    </div>
    </div>
Line 292: Line 308:
  <div class="step-tile">
  <div class="step-tile">
  <h2 class="step-title">
  <h2 class="step-title">
-
mini-Prep
+
Mini-Prep
</h2>
</h2>
  </div>
  </div>
Line 305: Line 321:
      </ul>
      </ul>
      <div class="step-recipe">
      <div class="step-recipe">
-
P<sub>tet</sub>-B0034-mRFP-dT on pSB6A1
+
R0040-B0032-E1010-B0015(on pSB6A1)
</div>
</div>
    </div>
    </div>
Line 323: Line 339:
   <div id="footer-content">
   <div id="footer-content">
<div id="footer-logo">
<div id="footer-logo">
-
<a href="http://igemhokkaidou.com/"><img style="height:150px;position:relative;" src="https://static.igem.org/mediawiki/2014/3/39/HokkaidoU_logo_transparent.png"></a>
+
<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 15:25, 9 September 2015

Notebook
Lab Documents

Ptet-B0032-mRFP-dT

  • Start

  • PCR

    R0040-B0034-E1010-B0015(on pSB6A1) SpeI_PstI_F Tm:72.8℃& Ptet_SpeI_R Tm:72.6℃ (vector)
    XbaI_B0032_mRFP_F Tm:76.3℃ & PstI_SpeI_R Tm:74.6℃ (insert)
    KOD Fx Neo

  • Gel extraction

    PCR products

  • Digestion

    Cut vector with SpeI, PstI (using 10×H buffer)
    Cut insert with XbaI, PstI (using 10×M buffer)

  • Dephosphorylation

    Antactic phosphorylate insert (using 10×Antarctic phosphatase buffer) 37℃ 35min 65℃ 10min

  • Ligation

    Ligate vector with insert

  • Transformation

    R0040-B0032-E1010-B0015(on pSB1A6)
    5.0µL DNA to DH5α

  • Colony PCR

    R0040-B0032-E1010-B0015(on pSB6A1) EcoI_XbaI_F Tm:70.9℃ & RBS_SpeI_R Tm:59.2℃

  • Culture

    R0040-B0032-E1010-B0015(on pSB6A1)

  • Mini-Prep

    R0040-B0032-E1010-B0015(on pSB6A1)

  • Complete!

Retrieved from "http://2014.igem.org/Team:HokkaidoU_Japan/Notebook/Pre_experiment/140910_Ptet-B0032-mRFP-dT"