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Team:HokkaidoU Japan/Notebook/asB0034/asB0034,32-construction
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| - | Ligate pSB1A3 | + | Ligate pSB1A3 with R0010-stem-asB0034-stem-B0015 |
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| - | Ligate pSB1A3 | + | Ligate pSB1A3 with R0010-stem-asB0032-stem-B0015 |
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Latest revision as of 15:29, 9 September 2015
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 with R0010-stem-asB0034-stem-B0015Ligate pSB1A3 with R0010-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!
