Team:Chiba/Calendar-Home/1 September 2008

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===Team:Output===
===Team:Output===
[[Team:Chiba/protocol/ligation/ligation|Ligation]]
[[Team:Chiba/protocol/ligation/ligation|Ligation]]
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*vector:[http://partsregistry.org/Part:BBa_R0010 BBa_R0010] insert:[http://partsregistry.org/Part:BBa_J52008 BBa_J52008]①
+
*vector:[http://partsregistry.org/Part:BBa_R0010 BBa_R0010] insert:[http://partsregistry.org/Part:BBa_J52008 BBa_J52008](1)
-
*negative control:[http://partsregistry.org/Part:BBa_R0010 BBa_R0010]②
+
*negative control:[http://partsregistry.org/Part:BBa_R0010 BBa_R0010](2)
<table width="250" border="4" cellpadding="0" cellspacing="0" bordercolor="#000000">
<table width="250" border="4" cellpadding="0" cellspacing="0" bordercolor="#000000">
<tr>
<tr>
<td width="257">Sample No.</td>
<td width="257">Sample No.</td>
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<td>①</td><td>②</td>
+
<td>1</td><td>②2/td>
</tr>
</tr>
<tr>
<tr>

Revision as of 20:55, 29 October 2008

>Home | Notebook

31 August 2008 <|> 2 September 2008

Contents

Laboratory work

Team:Input

  • Ptet＋RBS＋cIの、Mini Prep産物の濃度チェック。
  • -Ptet-cI-pMB1-Amp-の機能チェック。
  • -Ptet-cI-pMB1-Amp-,-PcI-GFP-p15A-Cm-をDouble Transformation(BWΔflic)
  • 結果-->GFPが発現していた。-->-PcI-GFP-p15A-Cm-が機能していないのでは？
  • ターミネーターをつける、pSB1A3にのせかえる。
  • -Ptet-cI-pMB1-Amp-のDigestion Check

混ぜ表

DoubleSingle
dH2O(μL) 12
XbaI(μL) 11
SpeI(μL) 1-
BSA(×10)(μL) 11
NEB(×10)(μL) 11
DNA(μL) 55
TOTAL(μL) 1010


UV irradiation test

  • two plasmids from ?
    • -Ptrc-LuxR-Plux-cI-colE1-Amp-
    • -PcI-GFP-p15a-Cm-の2plasmid(BW)
  1. Incubated cultures(from Glycerol Stocks) with 2mL of LB-Ampicillin Medium for 12 hours at 37 degrees.
  2. UV⊕:5.21,UV⊖:5.38<--?
  3. moved the cultures to small plates and started UV-irradiation.(wavelength:254nm,distance from the UV lamp to the cultures were 7.5cm.UV⊖のものは暗所に置いておく。)
  4. covered the plates with polyethylene wrap.
  5. after irradiation(Sample:0min,10min,30min,1h,2h,4h,6h,8h)(Negative Control:0h,1h,4h,8h)

よく攪拌してから、20μl採取。

  1. diluted each cultures with LB-ampicillin medium 104-fold and 105-fold (volume/volume).
  2. incoculated 20μl of the cultures and incubated for 12 hours at 37 degrees.
  3. counted the CFU(determined viable cell count).


Viable cell count

UV+ 104UV+ 105UV-104UV-105
0min 42374271156
10min 30151--
30min 1397--
1h 895104054
2h 51--
4h 2025450
6h 00--
8h 101155106

Team:Communication

(31/8)--->Gel Check
Chiba-0901.JPG
Sample No. 1~34
Sample DNA 2015
Loading Dye 43
TOTAL 24μl18μl
From left;
insert-1([http://partsregistry.org/Part:BBa_I9026 BBa_I9026])
insert-2([http://partsregistry.org/Part:BBa_I9030 BBa_I9030])
Chiba-0901-2.JPG
insert-3([http://partsregistry.org/Part:BBa_S03154 BBa_S03154])
Chiba-0901-3.JPG
vector-4([http://partsregistry.org/Part:BBa_R0010 BBa_R0010])
--->Gel extract
--->zymo
insert-1([http://partsregistry.org/Part:BBa_I9026 BBa_I9026]) -> 7μL
insert-2([http://partsregistry.org/Part:BBa_I9030 BBa_I9030]) -> 7μL
insert-3([http://partsregistry.org/Part:BBa_S03154 BBa_S03154]) -> 7μL
vector-4([http://partsregistry.org/Part:BBa_R0010 BBa_R0010]) -> 15μL
--->SAP
vector-4([http://partsregistry.org/Part:BBa_R0010 BBa_R0010])
--->Zymo
vector-4([http://partsregistry.org/Part:BBa_R0010 BBa_R0010]) -> 20μL


--->Gel Check
Chiba-0901-4.JPG
Sample DNA 1
Loading Dye 1
dH2O 4
TOTAL 6μl
From left;
  • insert-1([http://partsregistry.org/Part:BBa_I9026 BBa_I9026]) -> OK
  • insert-2([http://partsregistry.org/Part:BBa_I9030 BBa_I9030]) -> OK
  • insert-3([http://partsregistry.org/Part:BBa_S03154 BBa_S03154]) -> None --> Transformation [http://partsregistry.org/Part:BBa_S03154 BBa_S03154]-->

(2/9)Mini prepwith [http://partsregistry.org/Part:BBa_K084009 BBa_K084009], [http://partsregistry.org/Part:BBa_K084010 BBa_K084010]

  • vector-4([http://partsregistry.org/Part:BBa_R0010 BBa_R0010])


--->Ligation
Sample No. (1)(2)(3)(4)(5)
insert-1([http://partsregistry.org/Part:BBa_I9026 BBa_I9026]) 3-3--
insert-2([http://partsregistry.org/Part:BBa_I9030 BBa_I9030]) -3-3-
vector-4([http://partsregistry.org/Part:BBa_R0010 BBa_R0010]) 33--3
ligase 11111
Buffer 11111
dH2O 22555
TOTAL 10μl10μl10μl10μl10μl


--->Transformation
Competent cells : XL10GOLD 30μL
Transformed the following and grew on new ampicillin plates.
  1. [http://partsregistry.org/Part:BBa_K084009 BBa_K084009(Plac+RBS+RhlI+LVA, Amp)] -> 628 colonies
  2. [http://partsregistry.org/Part:BBa_K084010 BBa_K084010(Plac+RBS+CinI+LVA, Amp)] -> 500 colonies
  3. insert-1(RBS+RhlI+LVA) -> 9 colonies
  4. insert-2(RBS+CinI+LVA) -> No colonies on the plate
  5. vector-4(Plac, Amp) -> 186 colonies


--->(2/9) Colony PCR


Transformation

Competent cells : JW1908 40μL
Transformed the following and grew on new ampicillin plates.
  • [http://partsregistry.org/Part:BBa_K084007 BBa_K084007(Plac+RBS+LasI)]
  • [http://partsregistry.org/Part:BBa_K084008 BBa_K084008(Plac+RBS+RhlI)]
  • [http://partsregistry.org/Part:BBa_K084010 BBa_T9002(Ptet+RBS+LuxR+GFP)]
--->(2/9)Liquid Culture

Team:Output

Ligation

  • vector:[http://partsregistry.org/Part:BBa_R0010 BBa_R0010] insert:[http://partsregistry.org/Part:BBa_J52008 BBa_J52008](1)
  • negative control:[http://partsregistry.org/Part:BBa_R0010 BBa_R0010](2)
Sample No. 1②2/td>
vector 22
insert 30
Ligase Buffer 22
Ligase 11
dH2O 35
TOTAL 10μl10μl

-->R/T 2hour

Transformation

  • [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]
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