Team:Osaka/week12

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October 10 (Sun)

  1. Electrophoresis

dye 2μl, sample, ladder 5μl. 1% Agalose gel 50V 5min 100V 23min

  1. 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