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Team:HokkaidoU Japan/Notebook/asB0034/asE1010 Target gene vector Exchange to 6A1
From 2014.igem.org
Notebook
Lab Documents
E1010 Target gene vector exchange to pSB6A1
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Start
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PCR
R0040-B0034-E1010-B0015(on 6A1) EcoRI_XbaI_F Tm:74.5℃ & Ptet_SpeI_R Tm:72.6℃ (insert)KOD plus Neo -
Digestion
Cut vector with EcoRI, PstI (using 10×H buffer)Cut insert with EcoRI, PstI (using 10×M buffer) -
Gel extraction
digested vector & insert -
Dephosphorylation
Antarctic phosphorylate vector (using 10×Antarctic phosphatase buffer) 37℃ 35min 65℃ 10min -
Ligation
Ligate vector with insert -
Transformation
ligation productseach of 5.0µL to DH5α -
PCR
R0040-B0034-E1010-B0015(on 6A1) EcoRI_XbaI_F Tm:74.5℃ & Ptet_SpeI_R Tm:72.6℃ (insert) KOD plus Neo -
Digestion
Cut vector with EcoRI, PstI (using 10×H buffer)Cut insert with EcoRI, PstI (using 10×M buffer) -
Gel extraction
digested insert -
Ethanol Precipitation
digested insert -
Digestion
Cut vector with EcoRI, PstI (using 10×H buffer) -
Dephosphorylation
Antactic phosphorylate vector (using 10×Antarctic phosphatase buffer) 37℃ 35min 65℃ 10min -
Ligation
Ligate vector with insert -
Transformation
ligation products5.0µL DNA to DH5α -
Colony PCR
R0040-B0032-E1010-B0015(on pSB4C5) Ex_F Tm:70.4℃ & mRFP_R Tm:76.0℃R0040-B0034-E1010-B0015(on pSB4C5) Ex_F Tm:70.4℃ & mRFP_R Tm:76.0℃R0040-B0032-E1010-B0015(on pSB2K3) Ex_F Tm:70.4℃ & mRFP_R Tm:76.0℃R0040-B0034-E1010-B0015(on pSB2K3) Ex_F Tm:70.4℃ & mRFP_R Tm:76.0℃Kapa-Taq -
Liquid Culture
R0040-B0032-E1010-B0015(on pSB4C5)R0040-B0034-E1010-B0015(on pSB4C5)R0040-B0032-E1010-B0015(on pSB2K3)R0040-B0034-E1010-B0015(on pSB2K3) -
Mini-Prep
R0040-B0032-E1010-B0015(on pSB4C5)]R0040-B0034-E1010-B0015(on pSB4C5)R0040-B0032-E1010-B0015(on pSB2K3)R0040-B0034-E1010-B0015(on pSB2K3) -
Complete!
