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Team:HokkaidoU Japan/Notebook/Pre experiment/140910 Ptet-B0032-mRFP-dT
From 2014.igem.org
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R0040-B0034-E1010-B0015(on pSB6A1) SpeI_PstI_F Tm:72.8℃& Ptet_SpeI_R Tm:72.6℃ (vector) | R0040-B0034-E1010-B0015(on pSB6A1) SpeI_PstI_F Tm:72.8℃& Ptet_SpeI_R Tm:72.6℃ (vector) | ||
| - | + | </div> | |
| - | + | <div class="step-recipe"> | |
| + | XbaI_B0032_mRFP_F Tm:76.3℃ & PstI_SpeI_R Tm:74.6℃ (insert) | ||
| + | </div> | ||
| + | <div class="step-recipe"> | ||
| + | KOD Fx Neo | ||
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| - | + | Cut vector with SpeI, PstI (using 10×H buffer) | |
| - | + | </div> | |
| - | + | <div class="step-recipe"> | |
| + | Cut insert with XbaI, PstI (using 10×M buffer) | ||
</div> | </div> | ||
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| - | + | R0040-B0032-E1010-B0015(on pSB1A6) | |
| - | + | </div> | |
| + | <div class="step-recipe"> | ||
| + | 5.0µL DNA to DH5α | ||
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Revision as of 03:48, 18 October 2014
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!
