Team:Osaka/Notebook

From 2010.igem.org

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===August 16 (Mon)===
===August 16 (Mon)===
 +
# Restriction digests
 +
#* 1-13D,　1-2M,　1-1D,　1-18F with EcoRI, PstI to check for point mutation(s) affecting XbaI site
 +
#* 2-20J with XbaI, PstI to check/confirm XbaI activity
 +
# Gel electrophoresis of digests
 +
#* (RESULTS?)
 +
# Colony pick-up & transfer to solution culture (repeat; 2 each): 1-13D,　1-2M,　1-1D,　1-18F
 +
#* 3ml LB liquid medium; Amp 3μl or Kan 0.6μl added
 +
 +
===August 17 (Tue)===
 +
# Mini-prep of 1-13D,　1-2M,　1-1D,　1-18F parts inoculated yesterday (2 of each)
 +
# 'Cut check' (restriction digest + gel electrophoresis to confirm insert) of the miniprepped parts:
 +
#* 1-1D, 1-18F with EcoRI, SpeI
 +
#* 1-13D, 1-2M with XbaI, PstI
 +
#* (RESULTS?)
 +
# Transformation of 2-22P, 1-2J using 25μl of competent cells each
 +
 +
===August 18 (Wed)===
 +
iGEM Japan Meet-Up in Kyoto
 +
Attendance: Nakamura, Yasumoto, Saka, Kakuda
 +
 +
===August 19 (Thu)===
 +
1.液培へ
 +
    Anp 2-22P,1-2J 37℃ 10:00~
 +
 +
2.電気泳動
 +
    dye 2μl,サンプル 10μl,Ladder 2μl,2% Agalose gel
 +
 +
100V 30min,EtBr 30min 60min 90min
 +
    Ladder,1-1D(1)(2) d,1-2M(1)(2) d,1-18F(1)(2) d,1-13D(1)(2) d
 +
 +
    dye 2μl,サンプル 10μl,Ladder 2μl,1% Agalose gel
 +
 +
100V 30min,EtBr 30min 60min
 +
    Ladder,1-1D
 +
 +
3.制限酵素処理
 +
    プラスミド:1-1D
 +
            SpeI処理        EcoRI処理        SpeI+EcoRI処理
 +
    プラスミド    2.5μl            2.5μl            2.5μl
 +
    EcoRI        0            1μl            1μl
 +
    SpeI        1μl            0            1μl
 +
    H2O        41μl            41μl            40μl
 +
    10xNEbuffer2    5μl            5μl            5μl
 +
    100xBSA    0.5μl            0.5μl            0.5μl
 +
    total        50μl            50μl            50μl
 +
 
 +
    37℃ incubate 20:00~
 +
 +
4.mini prep プラスミド回収
 +
    2-22P,1-2J
 +
 +
8/20    中村、安本、坂
 +
1.電気泳動
 +
    dye 2μl,サンプル 10μl(1-1Dのみ 2μl),Ladder 2μl,1% Agalose gel
 +
 +
100V 20min,EtBr 30min
 +
    Ladder,1-1D Spe,1-1D Eco,1-1D Spe Eco,1-1D
 +
 +
2.制限酵素処理
 +
    2-22P,1-2J
 +
    プラスミド    2.5μl
 +
    d-H2O        40μl
 +
    10xNEbuffer    5μl
 +
    100xBSA    0.5μl
 +
    XbaI        1μl
 +
    PstI        1μl
 +
    total        50μl
 +
 +
    37℃ incubate
 +
 +
  電気泳動
 +
    dye 2μl,サンプル 10μl,Ladder 2μl,1% Agalose gel
 +
 +
100V 20min,EtBr 30min
 +
    Ladder,1-2J(1) d,1-2J(2) d,2-22P(2) d
 +
 +
    1-2J(1) d,1-2J(2) d,2-22P(2) d → 80℃,20min 失活
 +
 +
3. 制限酵素処理（サンプル2-20Jが見当たらなかったため）
 +
    8/9のレシピで再度制限酵素処理
 +
 +
  電気泳動
 +
    100V 20min,EtBr
 +
    2-20J d,Ladder
 +
    2-20J → 80℃,20min 失活
 +
 +
4.ライゲーション 3A assembly
 +
    制限処理したプラスミドを使用
 +
    2-20J                2μl    2-20J    　2μl
 +
    2-22P                2μl    1-2J(1)    　2μl
 +
    1-3A                2μl        　2μl
 +
    10xT4 DNA Ligasebuffer    2μl        　2μl
 +
    T4 DNA Ligase        1μl        　1μl
 +
    dH2O                11μl        　11μl
 +
    total                20μl        　20μl
 +
 +
    2-20H                2μl    2-20H    　2μl
 +
    2-22P                2μl    1-2J(1)    　2μl
 +
    1-3A                2μl        　2μl
 +
    10xT4 DNA Ligasebuffer    2μl        　2μl
 +
    T4 DNA Ligase        1μl        　1μl
 +
    dH2O                11μl        　11μl
 +
    total                20μl        　20μl
 +
 +
    room temprature 10min
 +
    80℃,20min 失活
 +
 
 +
5.トランスフォーメーション
 +
    8/17と同じ手順
 +
    コンピ(2) 50μl使用  DNA 2μl
 +
    クォーラロフェニコールPlateに150μlをまく
 +
    37℃ incubate 16:10~
 +
 +
8/21    中村、安本、坂
 +
1.液培へ
 +
    2-20J 2-22P(1)(2),2-20J 1-2J(1)(2)(3),2-20H 2-22P(1)(2),2-20H 1-2J(1)(2)(3)
 +
    Chr 1μl,LB 3ml
 +
    30℃ incubate 13:30~
 +
 +
2.ライゲーション
 +
    8/20と同じレシピ
 +
    1-1D,1-2M,1-3Aをライゲーション
 +
 +
3.トランスフォーメーション
 +
    SOC 10μl
 +
    37℃ 1h → 1.5h
 +
    Plate(c)にまく 16:40~ 37℃ incubate
 +
 +
 +
 +
 +
 +
 +
 +
 +
 +
 +
 +
 +
 +
 +
 +
 +
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Revision as of 10:03, 5 October 2010

July
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Week1 25 26 27 28 29 30 31
August
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Week3 8 9 10 11 12 13 14
Week4 15 16 17 18 19 20 21
Week5 22 23 24 25 26 27 28
Week6 29 30 31
September
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Week7 5 6 7 8 9 10 11
Week8 12 13 14 15 16 17 18
Week9 19 20 21 22 23 24 25
Week10 26 27 28 29 30
October
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Week10 1 2
Week11 3 4 5 6 7 8 9
Week12 10 11 12 13 14 15 16
Week13 17 18 19 20 21 22 23
Week14 24 25 26 27 28 29 30
31

Contents

Notebook

July 29 (Thu)

Attendance: Torigata, Takino, Teoh, Yasumoto, Kakuda, Saka, Tamura

  1. Safety lecture for junior members.
  2. Preparation of LB agar plates (26 Amp, 25 Kan, 25 Cam).

July 31 (Sat)

Attendance: Miyatake, Hirayama, Torigata, Teoh, Tadashi, Yasumoto, Kakuda, Saka

  1. Meeting
    • Summer project schedule
    • List of genes to clone

August 2 (Mon)

Attendance: Torigata, Takino, Teoh, Tadashi, Yasumoto, Saka

  1. Culture medium preparation
    • LB agar plates (49 antibiotic-less plates)
    • LB liquid medium (500 ml)
  2. Competent cells preparation - Nojima Method
    • SOB medium (MgCl2 not yet added) -> stored at 4˚C
    • TB buffer -> stored at 4˚C
    • Single-colony streaking of E. coli (strain DH5α) on 2 LB agar plates -> 37˚C incubation o/n

Tomorrow we shall prepare the remainder of the reagents and inoculate SOB with o/n-incubated E. coli.

August 3 (Tue)

Attendance: Nakamura, Kakuda, Saka, Yasumoto, Teoh

  1. Competent cells preparation (continued)
    • Preparation of glucose solution for making SOC medium.
    • Inoculation of pre-culture SOB from o/n-incubated agar plate colonies.
    • (Night) Transfer from pre-culture to growth culture.

August 4 (Wed)

Attendance: Nakamura, Saka, Kakuda, Yasumoto, Torigata, Teoh

  1. OD measurements throughout the day till required OD (0.3~0.7) was obtained.
  2. Completion of competent cells according to protocol.

August 5 (Thu)

Attendance: Nakamura, Yasumoto, Saka, Kakuda, Takino

  1. Transformation of Registry parts:
IDPart NameResistanceDescription
2-20J<bbpart>BBa_K118023</bbpart>AC. fermi endocellulase Cen A coding
2-20H<bbpart>BBa_K118022</bbpart>AC. fermi exocellulase Cex coding
1-2M<bbpart>BBa_B0034</bbpart>ARBS
1-13D<bbpart>BBa_B0010</bbpart>Aterminator
1-1D<bbpart>BBa_R0010</bbpart>Apromoter
1-18F<bbpart>BBa_E1010</bbpart>KRFP coding

Note: 'ID' is an internal identifier used by wet work members to simplify labeling etc. In the case of iGEM distribution parts it usually refers to the plate location.

August 6 (Fri)

Attendance: Nakamura, Saka, Yasumoto, Takino, Teoh

  1. Colony check
    • All transformed cells produced colonies!
    • Non-transformed cells (negative controls) did not grow on Amp, Kan or Cam plates -> confirmed lack of natural antibiotic resistance
  2. Colonies transferred to 3ml LB growth medium & incubated o/n at 37˚C

August 7 (Sat)

Attendance: Nakamura, Saka, Yasumoto, Takino

  1. Miniprep of o/n-incubated colonies (2-20J, 2-20H, 1-2M, 1-13D, 1-1D, 1-18F) using Sigma-Aldrich Plasmid Miniprep Kit
  2. Transformation of construction plasmids
IDPart NameResistanceDescription
1-1C<bbpart>pSB1A3</bbpart>Aconstruction plasmid containing mRFP coding device (<bbpart>BBa_J04450</bbpart>)
1-3A<bbpart>pSB1C3</bbpart>C(" ")
1-5A<bbpart>pSB1K3</bbpart>K(" ")
  1. Meeting

August 8 (Sun)

Attendance: Nakamura, Yasumoto

  1. Colony check
    • All parts successfully transformed
  2. Transfer to LB culture medium

August 9 (Mon)

  1. Miniprep of 1-1C
  2. Restriction digests of 2-20H, 2-20J, 1-1D, 1-18F, 1-1C, 1-13D, 1-2M
  3. Gel electrophoresis of digests ("cut check")
    • 2-20H, 2-20J, 1-1C -> OK
    • 1-1D, 1-18F, 1-13D, 1-2M -> not cut (single band, MW approx. equal to vector + insert)
  4. Transfer of 1-3A, 1-5A colonies to solution culture (repeat)
    • Yesterday's inoculated culture mediums contained the wrong antibiotics!
  5. Transformation of <bbpart>BBa_I13521</bbpart>, <bbpart>BBa_I13522</bbpart>, <bbpart>BBa_I13600</bbpart>, <bbpart>BBa_K204031</bbpart>, <bbpart>BBa_K204051</bbpart>, <bbpart>BBa_K204032</bbpart> from last year's stock

August 10 (Tue)

  1. Miniprep of 1-3A, 1-5A
  2. Restriction digests of 1-3A, 1-5A
  3. Gel electrophoresis
    1. 1st run: 1-3A, 1-5A (newly miniprepped), 1-1D, 1-18F, 1-13D, 1-2M (repeat)
      • 1-3A, 1-5A -> OK; others -> not cut (again)
    2. 2nd run: 1-1D, 1-18F, 1-13D, 1-2M (2nd repeat)
      • all parts not cut

August 11 (Wed)

  1. Miniprep of last year's parts transformed on Monday
  2. Gel electrophoresis of 1-1D, 1-18F, 1-13D, 1-2M (3rd repeat!!!)
    • all 4 not cut... AGAIN
    • so far all restriction digests involving XbaI seem to have failed; problem with enzyme stock?
    • will try with different set of restriction enzymes next week
  3. Sent miniprepped last year's parts to ECUST team in Shanghai, China

August 16 (Mon)

  1. Restriction digests
    • 1-13D,　1-2M,　1-1D,　1-18F with EcoRI, PstI to check for point mutation(s) affecting XbaI site
    • 2-20J with XbaI, PstI to check/confirm XbaI activity
  2. Gel electrophoresis of digests
    • (RESULTS?)
  3. Colony pick-up & transfer to solution culture (repeat; 2 each): 1-13D,　1-2M,　1-1D,　1-18F
    • 3ml LB liquid medium; Amp 3μl or Kan 0.6μl added

August 17 (Tue)

  1. Mini-prep of 1-13D,　1-2M,　1-1D,　1-18F parts inoculated yesterday (2 of each)
  2. 'Cut check' (restriction digest + gel electrophoresis to confirm insert) of the miniprepped parts:
    • 1-1D, 1-18F with EcoRI, SpeI
    • 1-13D, 1-2M with XbaI, PstI
    • (RESULTS?)
  3. Transformation of 2-22P, 1-2J using 25μl of competent cells each

August 18 (Wed)

iGEM Japan Meet-Up in Kyoto Attendance: Nakamura, Yasumoto, Saka, Kakuda

August 19 (Thu)

1.液培へ 

   Anp 2-22P,1-2J 37℃ 10:00~

2.電気泳動 

   dye 2μl,サンプル 10μl,Ladder 2μl,2% Agalose gel

100V 30min,EtBr 30min 60min 90min 

   Ladder,1-1D(1)(2) d,1-2M(1)(2) d,1-18F(1)(2) d,1-13D(1)(2) d

   dye 2μl,サンプル 10μl,Ladder 2μl,1% Agalose gel

100V 30min,EtBr 30min 60min 

   Ladder,1-1D

3.制限酵素処理 

   プラスミド:1-1D
           SpeI処理        EcoRI処理        SpeI+EcoRI処理
   プラスミド    2.5μl            2.5μl            2.5μl
   EcoRI        0            1μl            1μl
   SpeI        1μl            0            1μl
   H2O        41μl            41μl            40μl
   10xNEbuffer2    5μl            5μl            5μl
   100xBSA    0.5μl            0.5μl            0.5μl
   total        50μl            50μl            50μl
  
   37℃ incubate 20:00~

4.mini prep プラスミド回収 

   2-22P,1-2J

8/20 中村、安本、坂 1.電気泳動 

   dye 2μl,サンプル 10μl(1-1Dのみ 2μl),Ladder 2μl,1% Agalose gel

100V 20min,EtBr 30min 

   Ladder,1-1D Spe,1-1D Eco,1-1D Spe Eco,1-1D

2.制限酵素処理 

   2-22P,1-2J
   プラスミド    2.5μl
   d-H2O        40μl
   10xNEbuffer    5μl
   100xBSA    0.5μl
   XbaI        1μl
   PstI        1μl
   total        50μl

   37℃ incubate

 電気泳動
   dye 2μl,サンプル 10μl,Ladder 2μl,1% Agalose gel

100V 20min,EtBr 30min 

   Ladder,1-2J(1) d,1-2J(2) d,2-22P(2) d

   1-2J(1) d,1-2J(2) d,2-22P(2) d → 80℃,20min 失活

3. 制限酵素処理（サンプル2-20Jが見当たらなかったため） 

   8/9のレシピで再度制限酵素処理

 電気泳動
   100V 20min,EtBr
   2-20J d,Ladder
   2-20J → 80℃,20min 失活

4.ライゲーション 3A assembly 

   制限処理したプラスミドを使用
   2-20J                2μl    2-20J    　2μl
   2-22P                2μl    1-2J(1)    　2μl
   1-3A                2μl        　2μl
   10xT4 DNA Ligasebuffer    2μl        　2μl
   T4 DNA Ligase        1μl        　1μl
   dH2O                11μl        　11μl
   total                20μl        　20μl

   2-20H                2μl    2-20H    　2μl
   2-22P                2μl    1-2J(1)    　2μl
   1-3A                2μl        　2μl
   10xT4 DNA Ligasebuffer    2μl        　2μl
   T4 DNA Ligase        1μl        　1μl
   dH2O                11μl        　11μl
   total                20μl        　20μl

   room temprature 10min
   80℃,20min 失活

5.トランスフォーメーション 

   8/17と同じ手順
   コンピ(2) 50μl使用  DNA 2μl
   クォーラロフェニコールPlateに150μlをまく
   37℃ incubate 16:10~

8/21 中村、安本、坂 1.液培へ 

   2-20J 2-22P(1)(2),2-20J 1-2J(1)(2)(3),2-20H 2-22P(1)(2),2-20H 1-2J(1)(2)(3)
   Chr 1μl,LB 3ml
   30℃ incubate 13:30~

2.ライゲーション 

   8/20と同じレシピ
   1-1D,1-2M,1-3Aをライゲーション

3.トランスフォーメーション 

   SOC 10μl
   37℃ 1h → 1.5h
   Plate(c)にまく 16:40~ 37℃ incubate


















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