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Team:HokkaidoU Japan/Notebook/asB0034/asE1010 1A3 Construction
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Latest revision as of 15:30, 9 September 2015
Notebook
Lab Documents
Anti-Sense E1010 on 1A3 Construction
-
Start
-
PCR
R0010-stem-asE1010-stem-B0015(on pSB1C3) EcoRI_XbaI_F Tm:74.5℃ & Ptet_SpeI_R(insert) Tm:72.6℃KOD Fx Neo -
Digestion
Cut vector with EcoRI, PstI (using 10×H buffer)Cut insert with EcoRI, PstI (using 10×M buffer) -
Dephosphorylation
Antactic phosphorylate pSB1A3 (using 10×Antarctic phosphatase buffer) 37°C 35min 65℃ 10min -
Ligation
Ligate pSB1A3 with R0010-stem-asE1010-stem-B0015 -
Transformation
R0010-stem-asE1010-stem-B0015(on pSB1A3)5.0µL to DH5α -
Colony PCR
R0010-stem-asE1010-stem-B0015(on pSB1A3) EcoI_XbaI_F Tm:74.5℃ & PstI_SpeI_R Tm:74.6℃Kapa-Taq -
Culture
R0010-stem-asE1010-stem-B0015(on pSB1A3) -
Mini-Prep
R0010-stem-asE1010-stem-B0015(on pSB1A3) -
Complete!
