Team:Osaka/Notebook

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Revision as of 11:23, 5 October 2010

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:

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

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
   • (WHICH ENZYMES?)
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; 3μl Amp or 0.6μl Kan solution 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 secretion tag parts using 25μl of competent cells each

August 18 (Wed)

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

August 19 (Thu)

1. Transfer of 2-22P, 1-2J to solution culture
2. Gel electrophoresis of digests from 'cut check' products from Tuesday
   • repeat run, but each digest together with undigested plasmid DNA)
   • 2% agarose gel instead of the usual 1%
   • (RESULTS?)
3. Gel electrophoresis of 1-1D digest only
   • (RESULT?)
4. Multiple restriction digests of 1-1D to check for problems at restriction sites
   • tried the following: EcoRI only; SpeI only; EcoRI + SpeI
5. Night: miniprep of 2-22P, 1-2J inoculated in the morning

August 20 (Fri)

1. Gel electrophoresis of 1-1D and its digests
   • (RESULTS?)
2. 'Cut check' of parts miniprepped the night before
   • both 2-22P & 1-2J cut with XbaI, PstI
   • enzyme inactivation at 80℃, 20min
   • (RESULTS?)
3. Restriction digest of 2-20J (WHICH ENZYMES?)
4. Ligation according to 3A assembly method: 2-20J or 2-20H + 2-22P or 1-2J; 1-3A as vector
   • reaction mixture: 2μl of each plasmid, 2μl ligase buffer, 1μl T4 DNA ligase, water to make 20μl total
   • reaction at room temperature for 10min; ligase inactivation at 80℃ for 20min
5. Transformation of ligated parts using 50μl of competent cells each; 2μl ligation product; 150μl Chloramphenicol spread on agar plates before inoculation with pre-incubation mix

August 21 (Sat)

1. Transfer of white (non-RFP) colonies from yesterday's transformation to 3ml LB liquid medium added with 1μl Cam solution
   • we later realized that the upstream and downstream parts had been mixed up in these ligations, so the ligation products (and these corresponding solution cultures) were discarded
2. 3A assembly ligation: 1-1D as upstream, 1-2M as downstream, 1-3A as vector
   • ligation product designated as <1>
   • same ligation mix composition as yesterday's
3. Transformation of <1> with pre-incubation for 1.5hr instead of 1hr, transformation mix inoculated onto Chloramphenicol plate

August 22 (Sun)

1. Transfer of <1> to culture solution; incubation at 30℃ (why??)
   • Transformation of the following parts:

August 23 (Mon)

1.液培へ

   (1),(2),(3),2-4A
   LB 3ml,Amp 3μl,Chl 1μl                ←濃度で表したほうがいいのでは？？
   incubate 37℃ 9:30~

2.mini prep プラスミド回収

   1-1D・1-2M(3)

3.制限酵素処理

   プラスミド 1-1D・1-2M(3)    2.5μl
   EcoRI            1μl
   SpeI            1μl
   H2O            40μl
   10xNEBuffer2        5μl
   100xBSA            0.5μl
   total            50μl

   37℃ incubate 10:30~

　電気泳動

   dye 2μl,サンプル 10μl(ネガコン 2μl),Ladder 2μl    1%Agalose gel,100V 20min,EtBr 30min
   Ladder,1-1D・1-2M d,1-1D・1-2M

4.液培へ

   1-1D・1-2M    3つ
   12:53~ 37℃ incubate

5.トランスフォーメーション

   2-2O,3-11I    Amp
   13:30~ 37℃

August 24 (Tue)

1.transformation結果

   2-2O    100以上
   3-11I    100以上
           →液培に

2.miniprep プラスミド回収

   F1,F2,F3,2-4A    (8/22にトラフォした(1)~(3)はF1~3に名前を変更しました。)

3.制限処理酵素

   プラスミド F1,F2,F3        2.5μl
   EcoRI            1μl
   SpeI            1μl
   H2O            40μl
   10xNEBuffer2        5μl
   100xBSA            0.5μl
   total            50μl

   37℃ incubate 11:30~

   F3をもう一つ行う→F3'とする
   プラスミド 1F        2.5μl
   EcoRI            1μl
   PstI            1μl
   H2O            40μl
   10xNEBuffer2        5μl
   100xBSA            0.5μl
   total            50μl

   37℃ incubate 12:53~

4.電気泳動

   dye 2μl,サンプル 10μl,Ladder 2μl    1% Agalose gel,100V 30min,EtBr 30min
   Ladder,F1 d,F2 d,F3 d

   dye 2μl,サンプル 10μl(ネガコン 2μl),Ladder 2μl    1% Agalose gel,100V 30min,EtBr 30min
   F3,F3' d,Ladder

5.液培へ

   F1    LB 3ml,Amp 3μ
   37℃ incubate 14:30~

6.グリセロールストック作り

   (1-1D,1-2M,1-3A)の培養液 200mlと50%グリセリン 100mlを混合し-80℃へ

August 25 (Wed)

1.miniprep プラスミド回収

   F1,3-11I,2-2O

2.制限酵素処理

   プラスミド F1,3-11I        2.5μl
   EcoRI            1μl
   SpeI            1μl
   H2O            40μl
   10xNEBuffer2        5μl
   100xBSA            0.5μl
   total            50μl

   37℃ incubate 10:45~

3.電気泳動

   dye 2μl,サンプル 10μl,Ladder 2μl    1% Agalose gel,100V 30min,EtBr 40min
   Ladder,3-11I d,F1 d