Team:Osaka/Notebook

From 2010.igem.org

Revision as of 10:03, 5 October 2010 by Shaothing (Talk | contribs)

(diff) ← Older revision | Latest revision (diff) | Newer revision → (diff)

Notebook

July 29 (Thu)

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

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

July 31 (Sat)

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

Meeting
- Summer project schedule
- List of genes to clone

August 2 (Mon)

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

Culture medium preparation
- LB agar plates (49 antibiotic-less plates)
- LB liquid medium (500 ml)
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

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

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

August 5 (Thu)

Attendance: Nakamura, Yasumoto, Saka, Kakuda, Takino

Transformation of Registry parts:

ID	Part Name	Resistance	Description
2-20J	<bbpart>BBa_K118023</bbpart>	A	C. fermi endocellulase Cen A coding
2-20H	<bbpart>BBa_K118022</bbpart>	A	C. fermi exocellulase Cex coding
1-2M	<bbpart>BBa_B0034</bbpart>	A	RBS
1-13D	<bbpart>BBa_B0010</bbpart>	A	terminator
1-1D	<bbpart>BBa_R0010</bbpart>	A	promoter
1-18F	<bbpart>BBa_E1010</bbpart>	K	RFP 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

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
Colonies transferred to 3ml LB growth medium & incubated o/n at 37˚C

August 7 (Sat)

Attendance: Nakamura, Saka, Yasumoto, Takino

Miniprep of o/n-incubated colonies (2-20J, 2-20H, 1-2M, 1-13D, 1-1D, 1-18F) using Sigma-Aldrich Plasmid Miniprep Kit
Transformation of construction plasmids

ID	Part Name	Resistance	Description
1-1C	<bbpart>pSB1A3</bbpart>	A	construction plasmid containing mRFP coding device (<bbpart>BBa_J04450</bbpart>)
1-3A	<bbpart>pSB1C3</bbpart>	C	(" ")
1-5A	<bbpart>pSB1K3</bbpart>	K	(" ")

Meeting

August 8 (Sun)

Attendance: Nakamura, Yasumoto

Colony check
- All parts successfully transformed
Transfer to LB culture medium

August 9 (Mon)

Miniprep of 1-1C
Restriction digests of 2-20H, 2-20J, 1-1D, 1-18F, 1-1C, 1-13D, 1-2M
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)
Transfer of 1-3A, 1-5A colonies to solution culture (repeat)
- Yesterday's inoculated culture mediums contained the wrong antibiotics!
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)

Miniprep of 1-3A, 1-5A
Restriction digests of 1-3A, 1-5A
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)

Miniprep of last year's parts transformed on Monday
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
Sent miniprepped last year's parts to ECUST team in Shanghai, China

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