Team:Chiba/Calendar-Home/2 September 2008

From 2008.igem.org

(Difference between revisions)
(→Team:Input)
(→Team:Input)
Line 15: Line 15:
::PrecA-RFPにUVを当てたものはいづれもRFPが目で確認できなかった。
::PrecA-RFPにUVを当てたものはいづれもRFPが目で確認できなかった。
-
#Incubated cultures(from Glycerol Stocks) with 2mL of LB-Ampicillin Medium  for 12 hours at 37 degrees.
 
-
OD:#UV⊕:5.21,UV⊖(negative control):5.38
 
-
#moved the cultures to small plates and started UV-irradiation.(wavelength:254nm,distance from the UV lamp to the cultures were 7.5cm.Put the negartive control in adark place.
 
-
#covered the plates with polyethylene wrap.
 
-
#after irradiation(Sample:0min,10min,30min,1h,2h,4h,6h,8h)(Negative Control:0h,1h,4h,8h), we agitate the culture and took 20μl.
 
-
よく攪拌してから、20μl採取。
 
-
#diluted each cultures with LB-ampicillin medium 10<sup>4</sup>-fold and 10<sup>5</sup>-fold (volume/volume).
 
-
#incoculated 20μl of the cultures and incubated for 12 hours at 37 degrees.
 
-
#counted the CFU(determined viable cell count).
 
===Team:Communication===
===Team:Communication===

Revision as of 02:06, 30 October 2008

>Home | Notebook

1 September 2008 <|> 3 September 2008

Contents

Laboratory work

Team:Input

UV irradiation test Incubated cultures (from Glycerol Stocks) with 2ml of LB-Ampicillin Medium for 12 hours at 37 degrees. Pre-incubated was plated so as to produce about 1000 colonies.(Ptet:1,Ptet-RFP:1,PrecA-RFP:3)

  • 新たなAmpプレート8枚(2.5cm,6.5cm用のそれぞれ30sec,1min,30min,1h用)に、撒いて37°C,12hたったコントロール用のPtet,Ptet-RFPのコロニーをニトロセルロースでうつしてはる。
  • PrecA-RFPのプレートにUVを照射する。UVからの距離は2.5cmと,6.5cmの2パターンで実験する。We put　Negative Control　of　Prec-RFP in a dark place.
  • UV照射後30sec,1min,30min,1h経過したらそれぞれ（2.5cmでUV当てたもの、6.5cmでUV当てたもの、コントロール用）のプレートからニトロセルロースでコロニーをうつしとり、あらかじめコントロールをはっておいたAmpプレートにはりつけた。
  • それぞれのプレートでUVが照射されてからどのくらいの時間でRFPが発現するのかを調べるためにUV照射してから、0min,10min,30min,1h,2h,3h,4h,5h,6h後にスキャナーで取り込んで色の変化を見る。
  • 結果
PrecA-RFPにUVを当てたものはいづれもRFPが目で確認できなかった。

Team:Communication

(31/8)--->Gel Check
Chiba-0902.JPG
Sample DNA 1
Loading Dye 1
dH2O 4
TOTAL 6μl
From left;
[http://partsregistry.org/Part:BBa_I9026 BBa_I9026] -> OK, 100/μL
[http://partsregistry.org/Part:BBa_I9030 BBa_I9030] -> OK, 50ng/μL
[http://partsregistry.org/Part:BBa_S03154 BBa_S03154] -> OK, 30ng/μL (too low for the ligation:1/9 )


(1/9)---> Colony PCR
Colony PCR of 8 colonies from ligation plates (1/9:(1)[http://partsregistry.org/Part:BBa_K084009 BBa_K084009](R1~R8),(2)[http://partsregistry.org/Part:BBa_K084010 BBa_K084010](C1~C8)) and one from control plate([http://partsregistry.org/Part:BBa_F2620 BBa_F2620](2007)).
DNA Template 1
dNTP mix 5
Foward Primer 0.3
Reverse Primer 0.3
DNA polymerase TAQ 0.5
Thermopol Buffer 3
dH2O 20.5
TOTAL 30μL


95°C,5min -> ( 95°C,1min -> 52°C,1min -> 72°C,1min )･･･25cycles -> 72°C,10min -> 6°C


--->Gel Check

Chiba-0902-2.JPG
Sample DNA 1
Loading Dye 1
dH2O 4
TOTAL 6μl
From left;
  • Plac+RBS+RhlI+LVA
R1 -> OK
R2 -> Bad
R3~R7 -> OK
R8 -> Bad
Chiba-0902-3.JPG
From left;
  • Plac+RBS+CinI+LVA
C1,C2 -> OK
C3 -> Bad
C4~C6 -> OK
Chiba-0902-4.JPG
From left;
  • Plac+RBS+CinI+LVA
C7,C8 -> OK
  • [http://partsregistry.org/Part:BBa_F2620 BBa_F2620](2007):Positive control -> OK


--->(3/9)Mini prep



(1/9)--->Liquid Culture

Cultured the following cells (2mL LB-Amp, at 37°C, 7 hours)
from transformed plates:
  • [http://partsregistry.org/Part:BBa_K084007 BBa_K084007](Plac+RBS+LasI, Competent Cells : JW1908)
  • [http://partsregistry.org/Part:BBa_K084008 BBa_K084008](Plac+RBS+RhlI, Competent Cells : JW1908)
  • [http://partsregistry.org/Part:BBa_T9002 BBa_T9002](Ptet+RBS+LuxR+GFP, Competent Cells : JW1908)
from Glycerol Stock:
  • [http://partsregistry.org/Part:BBa_S03623 BBa_S03623](Ptet+RBS+LuxI, Competent Cells : JW1908)

--->(3/9)Phenotype test


Transformation

Competent cells : XL10G 30μL
  • [http://partsregistry.org/Part:BBa_C0161 BBa_C0161](2007)
  • [http://partsregistry.org/Part:BBa_C0161 BBa_C0161](2006)
  • [http://partsregistry.org/Part:BBa_C0261 BBa_C0261](2007)
  • [http://partsregistry.org/Part:BBa_C0261 BBa_C0261](2006)

--->(4/9)Mini prep

Team:Output

TIME RESPONCE (Solid)

Colony PCR

  • [http://partsregistry.org/Part:BBa_R0010 BBa_R0010]+[http://partsregistry.org/Part:BBa_J52008 BBa_J52008]
Sample No. 1
culture 1
Fwd primer 1.5
Rev primer 1.5
Thermo pol Buffer 3
dNTP mix 3
Taq DNA pol (NEB) 0.2(1　unit)
dH2O 19.8
TOTAL 30μl


-->95°C 5 min -->(95°C 1min -->50°C　30sec -->72°C　1min)x25 -->72°C　10min


Mini prep

  • [http://partsregistry.org/Part:BBa_R0079 BBa_R0079]
  • [http://partsregistry.org/Part:BBa_R0071 BBa_R0071]
  • [http://partsregistry.org/Part:BBa_R0077 BBa_R0077]
  • [http://partsregistry.org/Part:BBa_R0078 BBa_R0078]
  • [http://partsregistry.org/Part:BBa_R0062 BBa_R0062]
Retrieved from "http://2008.igem.org/Team:Chiba/Calendar-Home/2_September_2008"