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Team:Osaka/week12
From 2010.igem.org
1. Electrophoresis dye 2μｌ, sample, ladder 5μｌ. 1% Agalose gel 50V 5min 100V 23min
2.Ligation 048(C)
R1 (ES) 10/3 6μl FcenA (XP) 10/1 2μl 1-3A(EP) 10/9 2μl 10×T4 DNA ligase Buffer 2μｌ T4 DNA ligase 0.5μｌ DSW 7.5μｌ total 20μｌ
060(C)
001-2 (ES) 9/27 2μl GeneⅢ(XP) 10/6 6μl 1-3A(EP) 10/9 2μl 10×T4 DNA ligase Buffer 2μｌ T4 DNA ligase 0.5μｌ DSW 7.5μｌ total 20μｌ
051(C)
R2 (ES) 10/3 6μl 022 (XP) 10/1 2μl 1-3A(EP) 10/9 2μl 10×T4 DNA ligase Buffer 2μｌ T4 DNA ligase 0.5μｌ DSW 7.5μｌ total 20μｌ
066(A)
3-2P (ES) 10/9 GE 4μl (gel extract) 026 (EX) 10/9 GE 4μl 10×T4 DNA ligase Buffer 2μｌ T4 DNA ligase 0.5μｌ DSW 9.5μｌ total 20μｌ
069(A)
049 (ES) 10/9 GE 4μl 026 (EX) 10/9 GE 4μl 10×T4 DNA ligase Buffer 2μｌ T4 DNA ligase 0.5μｌ DSW 9.5μｌ total 20μｌ
070(A)
052 (ES) 10/9 GE 4μl 026 (EX) 10/9 GE 4μl 10×T4 DNA ligase Buffer 2μｌ T4 DNA ligase 0.5μｌ DSW 9.5μｌ total 20μｌ
16℃ 1h ,80℃ 20min
3.mini prep 058①,051②,016①,013②
4.Treatment of restriction enzyme cut check (check and assembly) 016① 10/10 15μｌ Xba I 0.5μｌ Pst I 0.5μｌ 10×NEbuffer2 5μｌ 100×BSA 0.5μｌ H2O 28.5μｌ total 50μｌ
plasmid 15μｌ
EcoR I 0.5μｌ Spe I 0.5μｌ 10×NEbuffer2 5μｌ 100×BSA 0.5μｌ H2O 28.5μｌ total 50μｌ
058①,013②,051②,1-13D,050①,025②
dye 2μｌ, sample 10μｌ, ladder 5μｌ. 1% Agalose gel 100V
5. Cel5B 3/4 α-agultinin PCR for DNA segment thermal cycle 94℃ 15sec 50℃ 30sec 74℃ 30sec 2cycle ↓add flanking primer 94℃ 15sec 62℃ 30sec 68℃ 1min 35cycle 72℃ 10min 4℃ ∞
5xPhusion HF Buffer 5μｌ
10mM dNTP 0.4μｌ
tube 3 0.2μｌ
Cel5B 1/2 0.2μｌ
primer rev 0.2μｌ
fwd2 0.2μｌ
phusion 0.2μｌ
DSW 13.6μｌ
total 20μｌ
α-agultinin PCR for DNA segment
98℃ 30sec 98℃ 10sec 72℃ 30sec 35cycle 72℃ 10min 4℃ ∞
5xPhusion HF Buffer 5μｌ
dNTPs 0.4μｌ
primer rev 0.2μｌ
fwd 0.2μｌ
phusion 0.2μｌ
DSW 14μｌ
total 20μｌ
dye 2μｌ, sample , ladder 5μｌ. 1% Agalose gel 100V 25min
6.To liquid culture 1-13D,1-12M,1-23L,013,056,069-2,061 →culture 37℃ incubate 1:00~
7.Transformation 10/10 ligation 048,060,051,066,069,070,1-13D compitent cell ①50μl＋DNA2μl 37℃ incubate 2:55~
8.PCR Products(ADH2,ENO1) treatment of restriction enzyme PCR Products 15μｌ EcoR I 0.5μｌ Spe I 0.5μｌ 10×NEbuffer2 5μｌ 100×BSA 0.5μｌ H2O 28.5μｌ total 50μｌ
PCR Products(d) 4μl 1-3A(ES,BB) 4μl 10×T4 DNA ligase Buffer 2μｌ T4 DNA ligase 0.5μｌ DSW 11.5μｌ total 20μｌ
Man→034 glr→031 ADH2→027 ENO→029 Cel44A→075
