Team:Osaka/week12

From 2010.igem.org

(Difference between revisions)
(New page: 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 ...)
(→October 16 (Sat))
 
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-
1. Electrophoresis
+
__NOTOC__
-
dye 2μｌ, sample, ladder 5μｌ. 
+
-
1% Agalose gel  50V 5min  100V 23min
+
-
2.Ligation
+
==October 10 (Sun)==
-
048(C)
+
# Electrophoresis
-
  R1 (ES)   10/3                       6μl
+
#Ligation
-
  FcenA (XP) 10/1                    2μl
+
#*048(C) R1 + FcenA
-
  1-3A(EP)   10/9                      2μl
+
#*060(C) 001-2 + GeneⅢ
-
  10×T4 DNA ligase Buffer        2μｌ
+
#*051(C) R2 + 022
-
  T4 DNA ligase                        0.5μｌ
+
#*066(A) 3-2P + 026
-
  DSW                                      7.5μｌ
+
#*069(A) 049 + 026
-
  total                                        20μｌ
+
#*070(A) 052 + 026
 +
#mini prep
 +
#*058①,051②,016①,013②
 +
#cut check
 +
#*016①,058①,013②,051②,1-13D,050①,025②
 +
#*PCR Products(ADH2,ENO1)
 +
#*Man→034
 +
#*glr→031
 +
#*ADH2→027
 +
#*ENO→029
 +
#*Cel44A→075
-
060(C)
+
#PCR for DNA segment
-
   001-2  (ES)  9/27                  2μl
+
#*Cel5B 3/4   α-agultinin
-
  GeneⅢ(XP)  10/6                    6μl
+
#To liquid culture
-
  1-3A(EP)  10/9                      2μl
+
#*1-13D,1-12M,1-23L,013,056,069-2,061 →culture
-
  10×T4 DNA ligase Buffer        2μｌ
+
#Transformation
-
  T4 DNA ligase                        0.5μｌ
+
#*048,060,051,066,069,070,1-13D
-
  DSW                                      7.5μｌ
+
-
  total                                        20μｌ
+
-
051(C)
+
==October 11 (Mon)==
-
  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)
+
#Transformation
-
  3-2P (ES)   10/9 GE              4μl        (gel extract)
+
#*034,031,027,029,075
-
026 (EX)  10/9 GE                  4μl
+
#*014-2,021-2,022-2,025-2,047
-
  10×T4 DNA ligase Buffer        2μｌ
+
#mini prep
-
  T4 DNA ligase                        0.5μｌ
+
#*1-23L,1-12M,061①,064-2②,056①②,013①②
-
  DSW                                      9.5μｌ
+
#*1-13D,061②,064-2①  →　destruction
-
  total                                        20μｌ
+
#cut check
 +
#*1-12M,064-2②,013①,056①,061①,1-23L
 +
#To liquid culture
 +
#*plate 025(10/8),022(10/8) each 2 colony to culture
 +
#Electrophoresis  
 +
#*064  ok
 +
#*061    x
 +
#*056    x
 +
#*013    ok
 +
#Ligation
 +
#*048(C) R1 + FcenA
 +
#*051(C) R2 + FcenA
 +
#*053(C) R2 + 024
 +
#*059(C) 001-2 + ToR
 +
#*068(K) 050 + 026  
 +
#*070(A) 052 + 054
 +
#*072(K) 049 + 054
 +
#*076(A) 050 + 054
 +
#*077(A) 2-4A + R1
 +
#cutcheck
 +
#*R2 →XP
 +
#*1-23L→ E
 +
#To liquid culture
 +
#*1-13D①,070①②,066①②,069①②,060①②,048①②,051①②
 +
#Electrophoresis
 +
#*1-23L ok
 +
#*036② ok
 +
#Ligation
 +
#*086(C) 024 + 1-23L
 +
#*078(A) 2-4A + R2
 +
#*085(C) 023 + 1-23L
 +
#*084(K) 049 + 1-23L
 +
#*083(K) 3-2P + 1-23L
 +
#*082(K) 035 + 1-23L
 +
#*081(C) 022 + 1-23L
 +
#*079(C) 036 + 1-23L
-
069(A)
+
==October 12 (Tue)==
-
  049 (ES)   10/9  GE                4μl
+
#mini prep
-
  026 (EX)  10/9  GE                  4μl
+
#*1-13D,022-2①②,048①②,060①②,066①②,069①②,070①②
-
  10×T4 DNA ligase Buffer        2μｌ
+
#*051①→　destrustion
-
  T4 DNA ligase                        0.5μｌ
+
#Transformation
-
   DSW                                      9.5μｌ
+
#*047,063,069,0071,078,079,080,081,082,083,084,085
-
  total                                        20μｌ
+
#cutcheck
 +
#*1-13D,022-2,048,066,069,070,1-13D,060
 +
#Electrophoresis
 +
#*1-13D →x
 +
#*1-13   →x
 +
#*022-2 →x
 +
#*048    →OK
 +
#*060    →?
 +
#*066   →OK
 +
#*069    →OK
 +
#*070    →OK
 +
#To liquid culture
 +
#*034,029,027,019-2,022-2,025-2,021-2,031,075,051,072,070,068,053,059,077,076,048,060,1-23L
 +
#cutcheck
 +
#*1-23L,048
 +
#Electrophoresis
 +
#.Ligation
 +
#*030(C) SUC d + 1-3A
 +
#*087(A) 048 + 026
 +
#*088(A) 048 + 054
 +
#*089(C) 2-4A + 054
-
070(A)
+
==October 13 (Wed)==
-
052 (ES)  10/9  GE                4μl
+
#mini prep
-
026 (EX)  10/9  GE                  4μl
+
#*027,034,029,053,077,060,025①,059,075,021-2,019-2,022-2,068,076,070,072,1-23L
-
   10×T4 DNA ligase Buffer       2μｌ
+
#*048、025②→destruction
-
   T4 DNA ligase                        0.5μｌ
+
#*051→x
-
   DSW                                      9.5μｌ
+
#Treatment of restriction enzyme
-
  total                                        20μｌ
+
#*027,034,029,053,077,060,025①,059,075,1-23L,021-2,019-2,022-2,068,076,070,072
 +
#Transformation
 +
#*030,087,088,089,
 +
#.Electrophesis
 +
#*014-2   x
 +
#*021-2  ok
 +
#*022      x
 +
#*025       x
 +
#*027      x
 +
#*029        x
 +
#*034        x
 +
#*053        ok
 +
#*077        ok
 +
#*076        ok
 +
#*075        ok
 +
#*072      ?
 +
#*070        ok
 +
#*068   ok
 +
#*060   ?
 +
#*059    ?
 +
#*059d
 +
#.To liquid culture
 +
#*047①②,063,063,069,071,078,079①②,080,081,082,083,084,085
 +
#To liquid culture
 +
#*019-2②,021-2②,025-2②③,027②,029②,034②
 +
#Ligation
 +
#*095 2-4A + 072
 +
#*096 053 + 026
 +
#*097 053 + 054
 +
#*098 2-4A + 068
 +
#*099 075 + 026
 +
#*070 052 + 054
 +
#*103 1-12M + 1-23L
 +
#*093 006 + 1-23L
 +
#*094 007 + 1-23L
 +
#*091 009 + 1-23L
 +
#*102 0-12M + 026
 +
#*051 R2 + FcenA
 +
#*105 2-4A + 076
-
16℃  1h  ,80℃   20min
+
==October 14 (Thu)==
 +
#mini prep
 +
#*019-2,022-2②,025-2②,027,029,034,047①②,069,071,078,080,081,082,083
 +
#Transformation
 +
#Treatment of restriction enzyme
 +
#*cut check→R2,022-2②,027②,029②,078,*054①,019-2②,069,071,081,075,082,083,019-2②,039,060,080
 +
#*054①,019-2②,069,071,081,075,082,083,019-2②,039,060,080
 +
#*for Gel extract→047①②,025-2③,034,1-5A,1-1C,1-3A
 +
#Electrophoresis
 +
#*R2   ok
 +
#*019  ok
 +
#*060  ok
 +
#*059  ok
 +
#*071  ok
 +
#*069    ok
 +
#*059    ok
 +
#*078    ok
 +
#*029    ok
 +
#*027    ?
 +
#*022  ok
 +
#*075    ok
 +
#*080    x
 +
#*081    x
 +
#*082      x
 +
#*083      x
 +
#To liquid culture
 +
#Treatment of restriction enzyme
 +
#*039②,036①,036②,047①,
-
3.mini prep
+
==October 15 (Fri)==
-
058①,051②,016①,013②
+
#mini prep
 +
#*087①②,088①②,089①②,030①②,079①
 +
#*051①②,086,085,025-2②,031①～④
 +
#Treatment of restriction enzyme
 +
#*079①,087①,088①,089①,030①,030①
 +
#Ligation
 +
#*081(A,K) 022 + 081
 +
#*082(A,K) 035 + 1-23L
 +
#*083(A,K) 3-2P + 1-23L
 +
#*092(A,K) 011 + 1-23L
 +
#*101(A,K) 075 + 1-23L
 +
#*106(C) 2-4A + 064
 +
#*108(C) 076 + 075
 +
#*047(K) 039 + 036
 +
#Electrophoresis
 +
#*030    x
 +
#*089    ok
 +
#*087    ok
 +
#079    ok
 +
#*036    ok
 +
#*039    ok
 +
#*glr    ok
 +
#To liquid culture
 +
#*094,099,102,103,105,51②,085②,091,086,025-2④,098,095,051,093
 +
#*097  x
 +
#*096  x
 +
#Transformation
 +
#*081,082,083,092,101,047,108,047-2,039,036,106
 +
#Ligation
 +
#*031-2(C) glr + 1-3A
 +
#*100(C) 039 + 079
 +
#*100-2(A) 039 + 079
-
4.Treatment of restriction enzyme
+
==October 16 (Sat)==
-
cut check (check and assembly)
+
#Transformation
-
016① 10/10        15μｌ
+
#*031-2(C),100(C),100-2(A),BYP5013(A)
-
Xba I                    0.5μｌ
+
#Ligation
-
Pst I                    0.5μｌ
+
#*81(C) 2-4A + 087
-
10×NEbuffer2          5μｌ
+
#*52 2-4A + 088
-
100×BSA              0.5μｌ
+
#*098(C) 2-4A + 068
-
H2O                    28.5μｌ
+
#Mini prep
-
total                      50μｌ
+
#*051(new),085,086,091,093,094,095,099,102,103,105
 +
#Transformation
 +
#*120(C),121(C),098(C),2-4A(A)
 +
#Treatment of restriction enzyme
 +
#*2-4A①,051(new),085,086,099,102,103,091,093,094,095,105
 +
#Electrophoresis
 +
#*2-4A    ok
 +
#*103      x
 +
#*102    ok
 +
#*099    ok
 +
#*086      ok
 +
#*085      x
 +
#*051    x
 +
#culture
 +
#*101,083,047,092,081,082,106,108,039,036,098,025-2,082,108,039,036
 +
#Electrophoresis
 +
#*091  x
 +
#*093    ok
 +
#*094    x
 +
#*095    x
 +
#*105    x
 +
#mini prep
 +
#*036①,079①
-
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. 
+
[[Team:Osaka/Notebook|Back to Notebook]]
-
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
+

Latest revision as of 21:04, 27 October 2010


October 10 (Sun)

  1. Electrophoresis
  2. Ligation
    • 048(C) R1 + FcenA
    • 060(C) 001-2 + GeneⅢ
    • 051(C) R2 + 022
    • 066(A) 3-2P + 026
    • 069(A) 049 + 026
    • 070(A) 052 + 026
  3. mini prep
    • 058①,051②,016①,013②
  4. cut check
    • 016①,058①,013②,051②,1-13D,050①,025②
    • PCR Products(ADH2,ENO1)
    • Man→034
    • glr→031
    • ADH2→027
    • ENO→029
    • Cel44A→075
  1. PCR for DNA segment
    • Cel5B 3/4 α-agultinin
  2. To liquid culture
    • 1-13D,1-12M,1-23L,013,056,069-2,061 →culture
  3. Transformation
    • 048,060,051,066,069,070,1-13D

October 11 (Mon)

  1. Transformation
    • 034,031,027,029,075
    • 014-2,021-2,022-2,025-2,047
  2. mini prep
    • 1-23L,1-12M,061①,064-2②,056①②,013①②
    • 1-13D,061②,064-2① →　destruction
  3. cut check
    • 1-12M,064-2②,013①,056①,061①,1-23L
  4. To liquid culture
    • plate 025(10/8),022(10/8) each 2 colony to culture
  5. Electrophoresis
    • 064 ok
    • 061 x
    • 056 x
    • 013 ok
  6. Ligation
    • 048(C) R1 + FcenA
    • 051(C) R2 + FcenA
    • 053(C) R2 + 024
    • 059(C) 001-2 + ToR
    • 068(K) 050 + 026
    • 070(A) 052 + 054
    • 072(K) 049 + 054
    • 076(A) 050 + 054
    • 077(A) 2-4A + R1
  7. cutcheck
    • R2 →XP
    • 1-23L→ E
  8. To liquid culture
    • 1-13D①,070①②,066①②,069①②,060①②,048①②,051①②
  9. Electrophoresis
    • 1-23L ok
    • 036② ok
  10. Ligation
    • 086(C) 024 + 1-23L
    • 078(A) 2-4A + R2
    • 085(C) 023 + 1-23L
    • 084(K) 049 + 1-23L
    • 083(K) 3-2P + 1-23L
    • 082(K) 035 + 1-23L
    • 081(C) 022 + 1-23L
    • 079(C) 036 + 1-23L

October 12 (Tue)

  1. mini prep
    • 1-13D,022-2①②,048①②,060①②,066①②,069①②,070①②
    • 051①→　destrustion
  2. Transformation
    • 047,063,069,0071,078,079,080,081,082,083,084,085
  3. cutcheck
    • 1-13D,022-2,048,066,069,070,1-13D,060
  4. Electrophoresis
    • 1-13D →x
    • 1-13 →x
    • 022-2 →x
    • 048 →OK
    • 060 →?
    • 066 →OK
    • 069 →OK
    • 070 →OK
  5. To liquid culture
    • 034,029,027,019-2,022-2,025-2,021-2,031,075,051,072,070,068,053,059,077,076,048,060,1-23L
  6. cutcheck
    • 1-23L,048
  7. Electrophoresis
  8. .Ligation
    • 030(C) SUC d + 1-3A
    • 087(A) 048 + 026
    • 088(A) 048 + 054
    • 089(C) 2-4A + 054

October 13 (Wed)

  1. mini prep
    • 027,034,029,053,077,060,025①,059,075,021-2,019-2,022-2,068,076,070,072,1-23L
    • 048、025②→destruction
    • 051→x
  2. Treatment of restriction enzyme
    • 027,034,029,053,077,060,025①,059,075,1-23L,021-2,019-2,022-2,068,076,070,072
  3. Transformation
    • 030,087,088,089,
  4. .Electrophesis
    • 014-2 x
    • 021-2 ok
    • 022 x
    • 025 x
    • 027 x
    • 029 x
    • 034 x
    • 053 ok
    • 077 ok
    • 076 ok
    • 075 ok
    • 072  ?
    • 070 ok
    • 068 ok
    • 060  ?
    • 059  ?
    • 059d
  5. .To liquid culture
    • 047①②,063,063,069,071,078,079①②,080,081,082,083,084,085
  6. To liquid culture
    • 019-2②,021-2②,025-2②③,027②,029②,034②
  7. Ligation
    • 095 2-4A + 072
    • 096 053 + 026
    • 097 053 + 054
    • 098 2-4A + 068
    • 099 075 + 026
    • 070 052 + 054
    • 103 1-12M + 1-23L
    • 093 006 + 1-23L
    • 094 007 + 1-23L
    • 091 009 + 1-23L
    • 102 0-12M + 026
    • 051 R2 + FcenA
    • 105 2-4A + 076

October 14 (Thu)

  1. mini prep
    • 019-2,022-2②,025-2②,027,029,034,047①②,069,071,078,080,081,082,083
  2. Transformation
  3. Treatment of restriction enzyme
    • cut check→R2,022-2②,027②,029②,078,*054①,019-2②,069,071,081,075,082,083,019-2②,039,060,080
    • 054①,019-2②,069,071,081,075,082,083,019-2②,039,060,080
    • for Gel extract→047①②,025-2③,034,1-5A,1-1C,1-3A
  4. Electrophoresis
    • R2 ok
    • 019 ok
    • 060 ok
    • 059 ok
    • 071 ok
    • 069 ok
    • 059 ok
    • 078 ok
    • 029 ok
    • 027  ?
    • 022 ok
    • 075 ok
    • 080 x
    • 081 x
    • 082 x
    • 083 x
  5. To liquid culture
  6. Treatment of restriction enzyme
    • 039②,036①,036②,047①,

October 15 (Fri)

  1. mini prep
    • 087①②,088①②,089①②,030①②,079①
    • 051①②,086,085,025-2②,031①～④
  2. Treatment of restriction enzyme
    • 079①,087①,088①,089①,030①,030①
  3. Ligation
    • 081(A,K) 022 + 081
    • 082(A,K) 035 + 1-23L
    • 083(A,K) 3-2P + 1-23L
    • 092(A,K) 011 + 1-23L
    • 101(A,K) 075 + 1-23L
    • 106(C) 2-4A + 064
    • 108(C) 076 + 075
    • 047(K) 039 + 036
  4. Electrophoresis
    • 030 x
    • 089 ok
    • 087 ok
  5. 079 ok
    • 036 ok
    • 039 ok
    • glr ok
  6. To liquid culture
    • 094,099,102,103,105,51②,085②,091,086,025-2④,098,095,051,093
    • 097 x
    • 096 x
  7. Transformation
    • 081,082,083,092,101,047,108,047-2,039,036,106
  8. Ligation
    • 031-2(C) glr + 1-3A
    • 100(C) 039 + 079
    • 100-2(A) 039 + 079

October 16 (Sat)

  1. Transformation
    • 031-2(C),100(C),100-2(A),BYP5013(A)
  2. Ligation
    • 81(C) 2-4A + 087
    • 52 2-4A + 088
    • 098(C) 2-4A + 068
  3. Mini prep
    • 051(new),085,086,091,093,094,095,099,102,103,105
  4. Transformation
    • 120(C),121(C),098(C),2-4A(A)
  5. Treatment of restriction enzyme
    • 2-4A①,051(new),085,086,099,102,103,091,093,094,095,105
  6. Electrophoresis
    • 2-4A ok
    • 103 x
    • 102 ok
    • 099 ok
    • 086 ok
    • 085 x
    • 051 x
  7. culture
    • 101,083,047,092,081,082,106,108,039,036,098,025-2,082,108,039,036
  8. Electrophoresis
    • 091 x
    • 093 ok
    • 094 x
    • 095 x
    • 105 x
  9. mini prep
    • 036①,079①


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Retrieved from "http://2010.igem.org/Team:Osaka/week12"