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Team:HokkaidoU Japan/Notebook/Length/as90 120
From 2014.igem.org
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| - | Cut | + | Cut as90(on H-stem) with NcoI,XhoI(using 10×Cut Smart) |
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Revision as of 03:28, 18 October 2014
Notebook
Lab Documents
Anti-sense 90, 120 Construction
-
Start
-
PCR & PCR purification
R0040-B0034-E1010-B0015as_RBS_XhoI_F Tm:68.2℃ & 90bp_asmRFP_Nco_R Tm:79.9℃as_RBS_XhoI_F Tm:68.2℃ & 120bp_asmRFP_Nco_R Tm:66.9℃ -
Digestion
Cut as90(on H-stem) with NcoI,XhoI(using 10×Cut Smart)Cut as120(on H-stem) with NcoI,XhoI(using 10×Cut Smart) -
Ethanol precipitation
as90, as120 -
Gel Extraction
H-stem -
Dephosphorylation
Antactic phosphorylate H-stem (using 10×Antarctic phosphatase buffer) 37℃ 35min 65℃ 10min -
Ligation
Ligate as90, as120 with H-stem(on pSB1A3) -
Transformation
as90, as120(on pSB1A3)5µL DNA to DH5α Turbo -
Colony PCR
as90 (on pSB1A3) XhoI_RBS_ Tm:68.2℃ & as90_NcoI_ Tm:79.9℃as120(on pSB1A3) XhoI_RBS_ Tm:68.2℃ & as120_NcoI_ Tm:66.9℃Kapa-Taq -
Liquid Culture
R0010-K1524104-B0015(on pSB1A3)R0010-K1524105-B0015(on pSB1A3) -
Mini-prep
R0010-K1524104-B0015(on pSB1A3)R0010-K1524105-B0015(on pSB1A3) -
Dye terminator
R0010-K1524104-B0015(on pSB1A3)R0010-K1524105-B0015(on pSB1A3) -
Complete!
