"
Team:HokkaidoU Japan/Notebook/Pre experiment/asmRFP
From 2014.igem.org
(Difference between revisions)
| Line 27: | Line 27: | ||
</li> | </li> | ||
</ul> | </ul> | ||
| - | <div class="step- | + | <div class="step-recipe"> |
asB0034-f Tm: 68.7°C asB0034-r Tm: 68.7°C | asB0034-f Tm: 68.7°C asB0034-r Tm: 68.7°C | ||
</div> | </div> | ||
| Line 48: | Line 48: | ||
</li> | </li> | ||
</ul> | </ul> | ||
| - | <div class="step- | + | <div class="step-recipe"> |
Cut pHN1257 with NcoI, XhoI(using 10×cut smart) | Cut pHN1257 with NcoI, XhoI(using 10×cut smart) | ||
</div> | </div> | ||
| Line 69: | Line 69: | ||
</li> | </li> | ||
</ul> | </ul> | ||
| - | <div class="step- | + | <div class="step-recipe"> |
digested pHN1257 | digested pHN1257 | ||
</div> | </div> | ||
| Line 90: | Line 90: | ||
</li> | </li> | ||
</ul> | </ul> | ||
| - | <div class="step- | + | <div class="step-recipe"> |
digested pHN1257 | digested pHN1257 | ||
</div> | </div> | ||
| Line 111: | Line 111: | ||
</li> | </li> | ||
</ul> | </ul> | ||
| - | <div class="step- | + | <div class="step-recipe"> |
Ligate pHN1257 with asB0034 | Ligate pHN1257 with asB0034 | ||
</div> | </div> | ||
| Line 132: | Line 132: | ||
</li> | </li> | ||
</ul> | </ul> | ||
| - | <div class="step- | + | <div class="step-recipe"> |
Ptrc-stem-asB0034-stem(pHN1257) on ice 30min each of 5.0µL DNA to DH5α 42°C 1min on ice 2min | Ptrc-stem-asB0034-stem(pHN1257) on ice 30min each of 5.0µL DNA to DH5α 42°C 1min on ice 2min | ||
</div> | </div> | ||
| Line 153: | Line 153: | ||
</li> | </li> | ||
</ul> | </ul> | ||
| - | <div class="step- | + | <div class="step-recipe"> |
Ptrc-stem-asB0034-stem(pHN1257) pHN1257_up_F Tm:60.1 asB0034-R Tm:68.7 | Ptrc-stem-asB0034-stem(pHN1257) pHN1257_up_F Tm:60.1 asB0034-R Tm:68.7 | ||
</div> | </div> | ||
| Line 174: | Line 174: | ||
</li> | </li> | ||
</ul> | </ul> | ||
| - | <div class="step- | + | <div class="step-recipe"> |
Ptrc-stem-asB0034-stem | Ptrc-stem-asB0034-stem | ||
</div> | </div> | ||
| Line 195: | Line 195: | ||
</li> | </li> | ||
</ul> | </ul> | ||
| - | <div class="step- | + | <div class="step-recipe"> |
Ptrc-stem-asB0034-stem | Ptrc-stem-asB0034-stem | ||
</div> | </div> | ||
| Line 216: | Line 216: | ||
</li> | </li> | ||
</ul> | </ul> | ||
| - | <div class="step- | + | <div class="step-recipe"> |
Ptrc-stem-asB0034-stem on pHN1257&Ptet-B0034-mRFP-dt on pSB6A1 on ice 30min each of 2.5µL DNA to DH5α 42C 30sec on ice 2min | Ptrc-stem-asB0034-stem on pHN1257&Ptet-B0034-mRFP-dt on pSB6A1 on ice 30min each of 2.5µL DNA to DH5α 42C 30sec on ice 2min | ||
</div> | </div> | ||
Revision as of 07:19, 25 September 2014
anti-sense mRFP Construction
-
Start
-
Primer Annealing
asB0034-f Tm: 68.7°C asB0034-r Tm: 68.7°C -
Digestion
Cut pHN1257 with NcoI, XhoI(using 10×cut smart) -
Gel extraction
digested pHN1257 -
Ethanol Precipitation
digested pHN1257 -
Ligation
Ligate pHN1257 with asB0034 -
Transformation
Ptrc-stem-asB0034-stem(pHN1257) on ice 30min each of 5.0µL DNA to DH5α 42°C 1min on ice 2min -
Colony PCR
Ptrc-stem-asB0034-stem(pHN1257) pHN1257_up_F Tm:60.1 asB0034-R Tm:68.7 -
Culture
Ptrc-stem-asB0034-stem -
mini-Prep
Ptrc-stem-asB0034-stem -
Transformation
Ptrc-stem-asB0034-stem on pHN1257&Ptet-B0034-mRFP-dt on pSB6A1 on ice 30min each of 2.5µL DNA to DH5α 42C 30sec on ice 2min -
Complete!
