Team:Chiba/Calendar-Home/21 August 2008

From 2008.igem.org

(Difference between revisions)
(New page: '''OUTPUT TEAM''' *transformation Lux CDABE(J32007:2007),RBS(B0034:2007) :結果:成功→RBS :失敗→Lux CDABE(二回行ったがどちらもコロニーができなかった)→三...)
(→Team:Communication)
 
(23 intermediate revisions not shown)
Line 1: Line 1:
-
'''OUTPUT TEAM'''
+
>[[Team:Chiba|Home]] | [[Team:Chiba/Notebook|Notebook]]
-
*transformation
+
-
Lux CDABE(J32007:2007),RBS(B0034:2007)
+
-
:結果:成功→RBS
+
[[Team:Chiba/Calendar-Home/20 August 2008|20 August 2008 <]]|[[Team:Chiba/Calendar-Home/22 August 2008|> 22 August 2008]]
-
:失敗→Lux CDABE(二回行ったがどちらもコロニーができなかった)→三回目に成功
+
 
 +
==Laboratory work==
 +
===Team:Input===
 +
(-->20/8)
 +
 
 +
Inoculated transformants for 12 hours in LB 2mL containing Ampicillin.
 +
 
 +
*[http://partsregistry.org/Part:BBa_R0051 BBa_R0051](2007)
 +
*[http://partsregistry.org/Part:BBa_R0051 BBa_R0051](2006)
 +
*[http://partsregistry.org/Part:BBa_J06650 BBa_J06650](2007)
 +
*[http://partsregistry.org/Part:BBa_J06650 BBa_J06650](2006)
 +
*[http://partsregistry.org/Part:BBa_J22136 BBa_J22136](2007)
 +
*[http://partsregistry.org/Part:BBa_J22141 BBa_J22141](2007)
 +
 
 +
BBa_J22141:After inoculated for 11 hours, added 20&mu;l IPTG and cultured one hour.
 +
 
 +
 
 +
*UV irradiation test  (plate phase)
 +
*two plasmids from(JW1908)
 +
**(Repressor)-Ptrc-LuxR-Plux-cI-colE1-Amp-
 +
**(Reporter)-PcI-GFP-p15a-Cm-
 +
 
 +
#Inoculated (Reporter) culture from glycerol stock(-80&deg;C freezer) in 2mL LB-Ampicillin, Chloramphenicol medium and LB-Ampicillin, Chloramphenicol, 100nmAHL mediumfor 12h, at 37&deg;C.
 +
#We diluted 10<sup>5</sup>-fold, and plated 20ul of the resulting solution to (LB-Amp,Cm)(LB-Amp,cm,AHL100nM) plates each other.
 +
#We cultured for 12h at 37&deg;C.
 +
#We iraddiated UV each plate. Wavelength:254nm,distance from the UV lamp to the cultures were 14cm.
 +
#We performed the same operations on plates following 9 or 21h of UV
 +
exposure.
 +
#Colonies from both plates were picked and cultured in 2mL of
 +
LB-Amp,Cm or LB-Amp, Cm, 100nMAHL for 12h at 37&deg;C.
 +
 
 +
#We diluted 10<sup>5</sup>-fold, and plated 20ul of the
 +
resulting solution to (LB-Amp,Cm)(LB-Amp,cm,AHL100nM) plates each other.
 +
 
 +
 
 +
*Colony count
 +
<table width="200" border="4" cellpadding="0" cellspacing="0" bordercolor="#000000">
 +
<td width="257"></td>
 +
<td>no AHL</td><td>AHL100nM</td>
 +
<tr>
 +
<td>9h</td>
 +
<td>606</td><td>453</td>
 +
</tr>
 +
<tr>
 +
<td>12h</td>
 +
<td>146</td><td>151</td>
 +
</tr>
 +
</table>
 +
result
 +
<BR>
 +
GFP fluorescence was observed from plates without AHL.
 +
 
 +
===Team:Communication===
 +
:(20/8)--->'''[[Team:Chiba/protocol/PCR|PCR]]'''
 +
::*[http://partsregistry.org/Part:BBa_C0070 BBa_C0070](2007)
 +
::*[http://partsregistry.org/Part:BBa_C0070 BBa_C0070](2006)
 +
::*[http://partsregistry.org/Part:BBa_C0076 BBa_C0076](2007)
 +
::*[http://partsregistry.org/Part:BBa_C0078 BBa_C0078](2007)
 +
::*[http://partsregistry.org/Part:BBa_C0078 BBa_C0078](2006)
 +
 
 +
 
 +
<table width="315" border="2" cellpadding="0" cellspacing="0" bordercolor="#000000">
 +
<tr>
 +
<td width="257">DNA Template</td>
 +
<td>1</td>
 +
</tr>
 +
<tr>
 +
<td>dNTP mix(&mu;L)</td>
 +
<td>10</td>
 +
</tr>
 +
<tr>
 +
<td>Foward Primer(&mu;L)</td>
 +
<td>5</td>
 +
</tr>
 +
<tr>
 +
<td>Reverse Primer(&mu;L)</td>
 +
<td>5</td>
 +
</tr>
 +
<tr>
 +
<td>DNA polymerase TAQ(&mu;L)</td>
 +
<td>1</td>
 +
</tr>
 +
<tr>
 +
<td>Thermopol Buffer(&mu;L)</td>
 +
<td>5</td>
 +
</tr>
 +
<tr>
 +
<td>dH<sub>2</sub>O(&mu;L)</td>
 +
<td>28</td>
 +
</tr>
 +
<tr>
 +
<td>TOTAL(&mu;L)</td>
 +
<td>50</td>
 +
</tr>
 +
</table>
 +
 
 +
 
 +
:95°C,5min -> ( 95°C,1min -> 52°C,1min -> 72°C,1min )･･･25cycles -> 72°C,10min -> 6°C
 +
 
 +
 
 +
 
 +
:--->'''[[Team:Chiba/protocol/gelcheck|Gel Check]]'''
 +
{|align="justify"
 +
|[[Image:Chiba-0821.JPG]]
 +
|
 +
:<table width="315" border="2" cellpadding="0" cellspacing="0" bordercolor="#000000">
 +
<tr>
 +
<td width="257">Sample DNA</td>
 +
<td>3</td>
 +
</tr>
 +
<tr>
 +
<td>Loading Dye(&mu;L)</td>
 +
<td>2</td>
 +
</tr>
 +
<tr>
 +
<td>dH<sub>2</sub>O(&mu;L)</td>
 +
<td>7</td>
 +
</tr>
 +
<tr>
 +
<td>TOTAL(&mu;L)</td>
 +
<td>12</td>
 +
</tr>
 +
</table>
 +
 
 +
:From left;
 +
::*[http://partsregistry.org/Part:BBa_C0078 BBa_C0078](2007) -> None
 +
::*[http://partsregistry.org/Part:BBa_C0078 BBa_C0078](2006) -> None
 +
::*[http://partsregistry.org/Part:BBa_C0076 BBa_C0076](2007) -> None
 +
::*[http://partsregistry.org/Part:BBa_C0070 BBa_C0070](2007) -> OK
 +
::*[http://partsregistry.org/Part:BBa_C0070 BBa_C0070](2006) -> OK
 +
|}
 +
 
 +
 
 +
 
 +
:'''[[Team:Chiba/protocol/transformation|Transformation]]'''
 +
:competent cells : XL10G
 +
::*[http://partsregistry.org/Part:BBa_S03154 BBa_S03154](2007)
 +
::*[http://partsregistry.org/Part:BBa_S03154 BBa_S03154](2006)
 +
::*[http://partsregistry.org/Part:BBa_I9026 BBa_I9026](2007)
 +
::*[http://partsregistry.org/Part:BBa_I9026 BBa_I9026](2006)
 +
::*[http://partsregistry.org/Part:BBa_I9030 BBa_I9030](2007)
 +
::*[http://partsregistry.org/Part:BBa_I9030 BBa_I9030](2006)
 +
 
 +
 
 +
 
 +
 
 +
--->(23/8)'''[[Team:Chiba/protocol/digestion|Digestion]]'''
 +
 
 +
===Team:Output===
 +
[[Team:Chiba/protocol/transformation|Transformation]]
 +
*[http://partsregistry.org/Part:BBa_J32007 BBa_J32007](2007)-->no colony-->no colony(2 times)-->colony(3times)
 +
*[http://partsregistry.org/Part:BBa_B0034 BBa_B0034](2007)-->colony

Latest revision as of 05:58, 30 October 2008

>Home | Notebook

20 August 2008 <|> 22 August 2008

Contents

Laboratory work

Team:Input

(-->20/8)

Inoculated transformants for 12 hours in LB 2mL containing Ampicillin.

  • [http://partsregistry.org/Part:BBa_R0051 BBa_R0051](2007)
  • [http://partsregistry.org/Part:BBa_R0051 BBa_R0051](2006)
  • [http://partsregistry.org/Part:BBa_J06650 BBa_J06650](2007)
  • [http://partsregistry.org/Part:BBa_J06650 BBa_J06650](2006)
  • [http://partsregistry.org/Part:BBa_J22136 BBa_J22136](2007)
  • [http://partsregistry.org/Part:BBa_J22141 BBa_J22141](2007)

BBa_J22141:After inoculated for 11 hours, added 20μl IPTG and cultured one hour.


  • UV irradiation test (plate phase)
  • two plasmids from(JW1908)
    • (Repressor)-Ptrc-LuxR-Plux-cI-colE1-Amp-
    • (Reporter)-PcI-GFP-p15a-Cm-
  1. Inoculated (Reporter) culture from glycerol stock(-80°C freezer) in 2mL LB-Ampicillin, Chloramphenicol medium and LB-Ampicillin, Chloramphenicol, 100nmAHL mediumfor 12h, at 37°C.
  2. We diluted 105-fold, and plated 20ul of the resulting solution to (LB-Amp,Cm)(LB-Amp,cm,AHL100nM) plates each other.
  3. We cultured for 12h at 37°C.
  4. We iraddiated UV each plate. Wavelength:254nm,distance from the UV lamp to the cultures were 14cm.
  5. We performed the same operations on plates following 9 or 21h of UV

exposure.

  1. Colonies from both plates were picked and cultured in 2mL of

LB-Amp,Cm or LB-Amp, Cm, 100nMAHL for 12h at 37°C.

  1. We diluted 105-fold, and plated 20ul of the

resulting solution to (LB-Amp,Cm)(LB-Amp,cm,AHL100nM) plates each other.


  • Colony count
no AHLAHL100nM
9h 606453
12h 146151

result
GFP fluorescence was observed from plates without AHL.

Team:Communication

(20/8)--->PCR
  • [http://partsregistry.org/Part:BBa_C0070 BBa_C0070](2007)
  • [http://partsregistry.org/Part:BBa_C0070 BBa_C0070](2006)
  • [http://partsregistry.org/Part:BBa_C0076 BBa_C0076](2007)
  • [http://partsregistry.org/Part:BBa_C0078 BBa_C0078](2007)
  • [http://partsregistry.org/Part:BBa_C0078 BBa_C0078](2006)


DNA Template 1
dNTP mix(μL) 10
Foward Primer(μL) 5
Reverse Primer(μL) 5
DNA polymerase TAQ(μL) 1
Thermopol Buffer(μL) 5
dH2O(μL) 28
TOTAL(μL) 50


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


--->Gel Check
Chiba-0821.JPG
Sample DNA 3
Loading Dye(μL) 2
dH2O(μL) 7
TOTAL(μL) 12
From left;
  • [http://partsregistry.org/Part:BBa_C0078 BBa_C0078](2007) -> None
  • [http://partsregistry.org/Part:BBa_C0078 BBa_C0078](2006) -> None
  • [http://partsregistry.org/Part:BBa_C0076 BBa_C0076](2007) -> None
  • [http://partsregistry.org/Part:BBa_C0070 BBa_C0070](2007) -> OK
  • [http://partsregistry.org/Part:BBa_C0070 BBa_C0070](2006) -> OK


Transformation
competent cells : XL10G
  • [http://partsregistry.org/Part:BBa_S03154 BBa_S03154](2007)
  • [http://partsregistry.org/Part:BBa_S03154 BBa_S03154](2006)
  • [http://partsregistry.org/Part:BBa_I9026 BBa_I9026](2007)
  • [http://partsregistry.org/Part:BBa_I9026 BBa_I9026](2006)
  • [http://partsregistry.org/Part:BBa_I9030 BBa_I9030](2007)
  • [http://partsregistry.org/Part:BBa_I9030 BBa_I9030](2006)



--->(23/8)Digestion

Team:Output

Transformation

  • [http://partsregistry.org/Part:BBa_J32007 BBa_J32007](2007)-->no colony-->no colony(2 times)-->colony(3times)
  • [http://partsregistry.org/Part:BBa_B0034 BBa_B0034](2007)-->colony
Retrieved from "http://2008.igem.org/Team:Chiba/Calendar-Home/21_August_2008"