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Team:HokkaidoU Japan/Notebook/Length/as90 120
From 2014.igem.org
(Difference between revisions)
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</ul> | </ul> | ||
<div class="step-recipe"> | <div class="step-recipe"> | ||
| - | R0040-B0034-E1010-B0015 | + | R0040-B0034-E1010-B0015 |
| + | as_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℃ | ||
| + | |||
</div> | </div> | ||
</div> | </div> | ||
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</ul> | </ul> | ||
<div class="step-recipe"> | <div class="step-recipe"> | ||
| - | Cut as 90, as120 | + | Cut as 90(on H-stem) with NcoI,XhoI(using 10×Cut Smart) |
| + | 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) |
| + | 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℃ |
| + | as120(on pSB1A3) XhoI_RBS_ Tm:68.2℃ & as120_NcoI_ Tm:66.9℃ | ||
| + | Kapa-Taq | ||
</div> | </div> | ||
| - | + | ||
| - | + | ||
| - | + | ||
</div> | </div> | ||
</div> | </div> | ||
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</ul> | </ul> | ||
<div class="step-recipe"> | <div class="step-recipe"> | ||
| - | + | R0010-K1524104-B0015(on pSB1A3) | |
| + | 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) | |
| + | 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) | |
| + | R0010-K1524105-B0015(on pSB1A3) | ||
| + | |||
</div> | </div> | ||
</div> | </div> | ||
Revision as of 01:13, 18 October 2014
Notebook
Lab Documents
Anti-sense 90, 120 Construction
-
Start
-
PCR & PCR purification
R0040-B0034-E1010-B0015 as_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 as 90(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!
