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Team:HokkaidoU Japan/Notebook/asB0034/asB0034,32-construction
From 2014.igem.org
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| - | Ligate pSB1A3 withR0010-stem-asB0034-stem-B0015 Ligate pSB1A3 withR0010-stem-asB0032-stem-B0015 | + | Ligate pSB1A3 withR0010-stem-asB0034-stem-B0015 |
| + | </div> | ||
| + | <div class="step-recipe"> | ||
| + | Ligate pSB1A3 withR0010-stem-asB0032-stem-B0015 | ||
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Revision as of 03:20, 18 October 2014
Notebook
Lab Documents
Anti-Sense B0034 (B0032) Construction
-
Start
-
Primer Annealing
asB0034_F Tm: 68.7℃ & asB0034_R Tm: 68.7℃asB0032_F Tm: 71.6℃ & asB0032_R Tm: 70.8℃ -
Digestion
Cut vector with NcoI, XhoI (using 10×cut smart buffer) -
Gel extraction
digested pSB1A3 -
Ethanol Precipitation
digested pSB1A3 -
Ligation
Ligate pSB1A3 withR0010-stem-asB0034-stem-B0015Ligate pSB1A3 withR0010-stem-asB0032-stem-B0015 -
Transformation
ligation products5.0µL to DH5α -
Colony PCR
R0010-stem-asB0034-stem-B0015(on pSB1A3) 100up_F Tm:62.8℃ & asB0034_R Tm:68.7℃R0010-stem-asB0032-stem-B0015(on pSB1A3) 100up_F Tm:74.5℃ & asB0032_R Tm:70.8℃Kapa-Taq -
Liquid Culture
R0010-stem-asB0034-stem-B0015(on pSB1A3)R0010-stem-asB0032-stem-B0015(on pSB1A3) -
Mini-Prep
R0010-stem-asB0034-stem-B0015(on pSB1A3)R0010-stem-asB0032-stem-B0015(on pSB1A3) -
Complete!
